
==== Front
Synth Syst Biotechnol
Synth Syst Biotechnol
Synthetic and Systems Biotechnology
2405-805X
KeAi Publishing

S2405-805X(24)00117-0
10.1016/j.synbio.2024.08.006
Original Research Article
Biosynthesis of the benzylpyrrolidine precursor in anisomycin by a unique ThDP-dependent enzyme
Qiao Yongjian
Wang Junbo
Zhang Dashan
Zheng Xiaoqing
Lin Baixin
Huang Yongkang
Liao Yulin
Deng Zixin
Kong Lingxin konglingxin7@sjtu.edu.cn
⁎⁎
You Delin dlyou@sjtu.edu.cn
⁎
State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, China
⁎ Corresponding author. dlyou@sjtu.edu.cn
⁎⁎ Corresponding author. konglingxin7@sjtu.edu.cn
22 8 2024
2025
22 8 2024
10 1 7685
12 6 2024
3 8 2024
19 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Anisomycin (compound 1), a multifunctional pyrrolidine antibiotic, primarily inhibits protein biosynthesis by binding to the ribosome. Upon binding to the ribosome, the para-phenol moiety of anisomycin inserts completely into the hydrophobic crevice of the A-site and blocks the access of the incoming aminoacyl-tRNAs, disrupting peptide bond formation. Hence, the para-methoxyphenyl group serves as a starting point for developing novel anisomycin analogs with potent antifungal and insecticidal properties. However, the activation and condensation mechanism of phenylpyruvic acid has not yet been elucidated. In this study, genetic manipulations of aniP and its homologue siAniP confirmed their indispensable role in 1 biosynthesis. Bioinformatics analysis suggested that AniP and siAniP function as transketolase. siAniP was found to catalyzed condensation between 4-hydroxyphenylpyruvic acid (3) and glyceraldehyde (GA), initiating pyrrolidine synthesis. siAniP was specific for aromatic keto acids and tolerant of aliphatic and aromatic aldehydes, and was able to catalyze the asymmetric intermolecular condensation of two keto acids, leading to the formation of 24 α-hydroxy ketone. To the best of our knowledge, siAniP is the first TK that catalyzes the transfer of a C2 ketol and symmetrical intermolecular coupling using aromatic keto acids as donor substrates. Structural analysis, docking model construction, and site-directed mutagenesis identified that I220, H275, R322 and W391 were crucial for substrate binding. Moreover, sequence similarity network (SSN)-based genome neighborhood network (GNN) analyses of AniP suggested the widespread occurrence of the AniP-like-mediated reaction in the biosynthesis of 1 and its analogs, particularly in the assembly of benzylpyrrolidine. These findings not only expand the repertoire of TKs but also provide a potent biocatalyst that could be used for the structural innovation of 1 and its derivatives.

Keywords

Transketolase
Aromatic keto acid
Promiscuity
Anisomycin
Biosynthesis
==== Body
pmc1 Introduction

Streptomyces species have been considered as a repository of diverse and valuable natural products [1]. Natural products produced by Streptomyces account for 70–80 % of all natural bioactive products with pharmacological or agrochemical applications [2]. Among them, pyrrolidines, pyrrolidine alkaloids, and pyrrolidine-based hybrid molecules are biologically and pharmacologically active compounds with diverse activities [3]. The well-known drugs containing a pyrrolidine ring include clemastine (antihistaminic) [4], glycopyrronium (anticholinergic) [5] and bepridil (antihypertensive) [6]. Recently, therapeutic molecules containing a pyrrolidine ring in their structures, such as daridorexant (for insomnia), pacritinib (a JAK-2 inhibitor), and futibatinib (a FGFR-4 inhibitor) were approved by the FDA in 2022 [7]. Their vital roles in pharmacotherapy make them a promising scaffold for developing more novel biologically active compounds and drug candidates.

Anisomycin (1, Fig. 1A), a pyrrolidine antibiotic, features a unique pyrrolidine structure with a trans-diol and exhibits diverse biological and pharmacological activities [8]. 1 was first isolated from S. roseochromogenes and S. griseolus in 1954 [9]. 1 is primarily recognized as a potent and reversible inhibitor of protein synthesis in eukaryotic organisms, owing to its reversible binding to the 60S ribosomal subunit [10]. It has been successfully used in clinics for the treatment of amoebic dysentery and trichomoniasis. Additionally, it is one of the important and effective components in Agricultural Antibiotic 120, which has been widely used for controlling crop diseases in China [8]. Moreover, 1 has been reported to be a multifunctional drug, playing an important role in modulating signal transduction pathways [11], regulating gene expression [12], inhibiting the consolidation of new memories [13], and suppressing the replication of viruses [14].Fig. 1 The biosynthesis of 1 and in vivo function verifications of aniP and its homologous gene siAniP. (A) The biosynthetic pathway of 1. AniP was proposed to catalyze the condensation between 3 and D-G3P to yield the key intermediate 4. (B) The BGC of 1 from S. hygrospinosus and the homologous BGC from S. inusitatus. (C) LC-MS analysis of 1 production in the genetic-interruption ΔaniP strain (III), the complementary strain ΔaniP::aniP(IV) and ΔaniP::sianiP(V), the wild-type (WT) strain (II). Standard 1 (I) is used as a control.

Fig. 1

Since its discovery, the diverse biological activities and structural features of 1 have attracted the attention of many chemists and biologists. Compared to the rapidly developed total synthesis of 1, the biosynthetic machinery has remained obscure for more than 60 years. In our previous work, the biosynthetic gene cluster of 1 from S. hygrospinosus var. beijingensis has been cloned using a bioactivity-guided library screening approach (Fig. 1B) [8]. The characterization of the short-chain dehydrogenase AniN revealed a distinct multistep pyrrolidine-forming reaction for the core benzylpyrrolidine scaffold construction [8]. By employing a combination of bioinformatic analysis, reverse genetics, chemical analysis, and in vitro biochemical assays, the biosynthetic machinery was revealed. The biosynthesis of 1 starts from the deamination of l-tyrosine by AniQ, yielding 4-hydroxyphenylpyruvic acid 3. Meanwhile, AniQ also catalyzes the second transamination of 4 to generate an amino group for pyrrolidine formation. Afterward, the pyrrolidine ring was acetylated by AniI, and the phenol moiety was methylated by AniK [15]. The tailoring reactions did not follow any particular sequence, and the tailoring enzymes can tolerate different substrates. Before the second transamination, an α-keto acid-incorporating transketolase was needed to catalyze the transketolase-type condensation reaction between 3 and glyceraldehyde-3-phosphate (G3P) to yield 4 (Fig. 1A). The condensation lays the foundation for benzylpyrrolidine scaffold construction, but the mechanism remains poorly understood.

In this study, in vivo genetic complementation of aniP and its homologue siAniP demonstrated their indispensable role in 1 biosynthesis. siAniP turned out to be a transketolase that catalyzed the condensation between 3 and d-glyceraldehyde (D-GA) rather than the classic phosphorylated G3P to form 4. Moreover, siAniP showed specificity for aromatic keto acids as donors but displayed promiscuity towards both aliphatic and aromatic aldehydes as acceptors. Structural analysis and site-directed mutagenesis suggested several amino acid residues crucial for reactivity, possibly by shaping the substrate-binding pocket and stabilizing thiamin diphosphate (ThDP). AniP-like proteins with high sequence identity are mainly distributed in candidate biosynthetic gene clusters (BGCs) encoding 1 and its analogs. The differences in BGCs arrangement could be tapped for other benzylpyrrolidine analogs. Our results fill the knowledge gap related to transketolase-type condensation during the biosynthesis of 1, expand the catalytic repertoire of transketolase, and offer an aromatic keto acid-specific biocatalyst that could be further developed to synthesize new derivatives.

2 Materials and methods

2.1 Strains, plasmids, primers, and general methods

The strains, plasmids and primers used in this study were listed in Tables S1 and Tables S2, respectively. All E. coli strains were cultivated in liquid Luria-Bertani (LB) medium or on solid LB agar plates at 37 °C. MS medium (20 % agar, 20 % mannitol, 20 % soya flour) was used for sporulation of Streptomyces strains. TSBY (30 % tryptone soya broth, 10 % yeast extract, 10.3 % sucrose) was used as the seed medium. For the production of 1, all Streptomyces strains were cultured at 30 °C in a fermentation medium consisting of 1 % corn starch, 2 % soluble starch, 1 % soya flour, 0.02 % KH2PO4, 0.3 % NaCl, 0.3 % NH4Cl, and 0.4 % CaCO3.

2.2 Construction of the E. coli-Streptomyces shuttle vector pIB139tsr

The DNA fragment covering the intact thiostrepton resistance gene in pJTU1278 of 1202 bp was amplified using primers tsr-F/tsr-R (Table S2). The resultant PCR product was cloned into NheI-digested plasmid pIB139 [16] using a one-step cloning kit (Vazyme Biotech Co., Ltd., Nanjing, China) to generate the derivative plasmid pIB139tsr.

2.3 Construction of gene complementary strains

To construct the ΔaniP complementary strains ΔaniP::aniP and ΔaniP::sianiP, the corresponding intact gene was amplified from the genomic DNA using primers aniP-139-F/aniP-139-R and sianiP-139-F/sianiP-139-R (Table S2), respectively. The PCR products were cloned into NdeI/XbaI-digested pIB139tsr using one-step cloning to generated plasmids pIB139tsr-aniP and pIB139tsr-sianiP, respectively. The positive verified plasmids were transferred into E. coli ET12567/pUZ8002 and introduced into the ΔaniP mutant via intergeneric conjugation following the standard procedure. The positive complementary strains were selected using thiostrepton and then verified via PCR using the primers aniP-139-F/aniP-139-R and sianiP-139-F/sianiP-139-R, respectively (Table S2).

2.4 Fermentation and analysis of 1

The Streptomyces spore stock was first inoculated into TSBY medium and cultured at 30 °C for approximately 48 h. The seed broth was then inoculated into the fermentation medium (5 % v/v) and incubated at 30 °C for another 5 days. After fermentation, the culture broth was extracted three times with an equal volume of ethyl acetate. The organic extract obtained was first dried using rotary evaporation and then redissolved in methanol before LC-MS analysis. The resulting product was analyzed using an UltiMate 3000 HPLC system connected to a LTQ XL™ Linear Ion Trap Mass Spectrometer (Thermo Fisher Scientific Inc.) with an Agilent ZORBAX SB-C18 column (5 μm, 4.6 × 250 mm). The column was equilibrated with 95 % (v/v) solvent A (H2O with 0.1 % (v/v) formic acid) and 5 % (v/v) solvent B (acetonitrile) and developed with a linear gradient (5–30 min, from 5 % B to 50 % B, 30–45 min, from 50 % B to 95 % B) and then kept 95 % B for 5 min at a flow rate of 0.5 ml/min with UV detection at 280 nm.

2.5 Bioinformatics analysis

The bioinformatics analysis of AniP was conducted using the online software NCBI BlastP (https://blast.ncbi.nlm.nih.gov/). Multiple-sequence alignment was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo) [17] and optimized using ESPript 3.0 (https://espript.ibcp.fr/ESPript/cgibin/ESPript.cgi) [18]. The phylogenetic tree was constructed by MEGA11 [19] using the neighbor-joining method and optimized by Chiplot (https://www.chiplot.online/) [20]. Proteins used for phylogenetic tree construction were listed in Table S3. The sequence similarity network (SSN) and genome neighborhoods analysis of AniP were conducted using enzyme similarity tool (https://efi.igb.illinois.edu/efi-est/) [21] and the SSN was visualized in Cytoscape 3.9.0 software using the ‘yFiles Organic’ layout.

2.6 Construction of protein expression plasmids and site-directed mutagenesis

To construct the plasmids for protein expression in E. coli, the aniP and sianiP genes were PCR amplified from the genomic DNA of S. hygrospinosus and S. inusitatus using primers aniP-28a-F/aniP-28a-R and sianiP-28a-F/sianip-28a-R, respectively. The PCR products were cloned into the corresponding sites of pET28a (+) and then transformed into E. coli DH10B. Site-directed mutagenesis of siAniP was performed using inverse PCR with the plasmid pET28a-sianiP as a template and the primers listed in Table S2. After digestion with DpnI, the PCR product was transformed into E. coli DH10B.

2.7 Proteins expression and purification

The expression plasmids for AniP, siAniP and site-directed mutant proteins were transformed into E. coli BL21 (DE3). The resultant E. coli BL21 cells were cultured at 37 °C and 220 r.p.m. on a shaking incubator in Luria-Bertani (LB) medium supplemented with kanamycin at a final concentration of 50 μg/ml until reaching an OD600 of 0.6. Isopropylthio-β-d-galactoside (IPTG) with a final concentration of 0.1 mM was added to the culture after cooling at 4 °C for 30 min to induce protein expression. The cells were further cultured at 16 °C for 20 h. Then, the cells were harvested by centrifugation (3500 rpm, 15 min, 4 °C) and resuspended in 25 ml of buffer A (50 mM Tris-HCl, pH 8.0, 0.3 M NaCl) and lysed by sonication for 40 min. Cellular debris was removed by centrifugation (15000 rpm, 60 min, 4 °C), and the supernatant was used to purify the protein by nickel-affinity chromatography. The protein was eluted with an increasing gradient of buffer B (500 mM imidazole in buffer A). Eluted proteins were concentrated using centrifugal filters (Amicon) with a 10 kDa cutoff, desalted with a PD-10 desalting column (GE Healthcare) and finally exchanged into buffer C (50 mM Tris-HCl, 100 mM NaCl, 10 % glycerol, pH 7.5). Size exclusion chromatography (SEC) was performed using a Superdex 200 10/300 GL column (GE Healthcare) at 4 °C equilibrated in buffer D (50 mM Tris-HCl, 100 mM NaCl, pH 7.5). Next, a 1-mL aliquot of the isolated protein (10 mg/mL) was loaded onto the column and eluted at a flow rate of 0.4 mL/min. The purified protein was stored in buffer C at −80 °C until further analysis. Protein concentration was determined using the Bradford assay with bovine serum albumin as a standard.

2.8 In vitro enzymatic assays of siAniP

A typical 100 μl system consists of 1 mM DTT, 10 mM MgCl2, 1 mM ThDP, 10 mM keto acid substrate, 10 mM aldehyde substrate or another different keto acid dissolved in 50 mM potassium phosphate buffer (pH 7.5). The reactions were initiated by adding siAniP (final concentration was 20 μM) and then incubated for 4 h at 30 °C. The reactions were stopped by adding 150 μL of methanol, followed by centrifugation at 13,000 rpm for 10 min. The supernatant was then subjected to LC-MS analysis using an Agilent 6546 LC/Q-TOF system following the same protocol as described above.

2.9 Kinetic analysis of siAniP towards 3

The assay was carried out in a 100 μl reaction system consists of 1 mM DTT, 10 mM MgCl2, 1 mM ThDP, 15 mM D-GA, 3 (varying from 50 μM to 15 mM) in 50 mM potassium phosphate buffer (pH 7.5). The reactions were initiated by addition of 2 μM siAniP, incubated at 30 °C for 30 min and quenched by adding methanol of 100 μL. Reactions were run in triplicate and the steady-state parameters kcat and Km were determined by nonlinear fitting of the Michaelis-Menten equation using GraphPad Prism 8.0.

3 Results

3.1 AniP is an essential ThDP-dependent transketolase for the biosynthesis of 1

The previous genetic deletion of aniP has completely abolished the production of 1 without the accumulation of any biosynthetic intermediates (Fig. 1C). To exclude other possible explanations of the phenotype of ΔaniP, a single copy of aniP on the integrative plasmid pIB139tsr under the control of a strong constitutive promoter permE* was transferred into the ΔaniP strain to construct the complementary strain Δanip:aniP (Fig. S1). The fermentation products of ΔaniP:aniP were analyzed by LC-MS, and the production of 1 was restored (Fig. 1C). This data confirmed the indispensability of aniP for 1 biosynthesis.

The aniP gene cluster encodes a transketolase (TK) with 603 amino acids, which belongs to the ThDP-dependent enzymes, as indicated by the NCBI BlastP analysis. TK typically catalyzes the reversible transfer of two-carbon units from donor ketose phosphates to acceptor aldose phosphates in a ThDP and divalent metal-dependent manner [22]. TK links the pentose phosphate pathway with glycolysis and is also involved in the biosynthesis of nucleic acids and aromatic amino acids. The phylogenetic analysis (Fig. S2) showed that AniP and its homologous proteins (with a sequence identity of 33–99 %) clustered into a small subclade. Meanwhile, AniP-like proteins occupied the same clade as another subgroup of TK namely 1-deoxy-d-xylulose 5-phosphate synthase (DXPS), but were far from those ThDP-dependent enzyme groups, including acetolactate synthase (ALS), indole-3-pyruvate decarboxylase (IpdC), benzoylformate decarboxylase (BFD), pyruvate decarboxylase (PDC), benzaldehyde lyase (BAL), 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase (MenD) and branched-chain alpha-ketoacid decarboxylase (KdcA). DXPS catalyzes the formation of DXP from pyruvate (donor) and D-G3P (acceptor) [23]. ThDP-dependent enzymes vary largely in sequence and domain organization, but they share two essential common domains for binding and activation of ThDP: the pyrophosphate (PP) and the pyrimidine (PYR) domain [24]. The PP domain contains a conserved GDX25-30N motif, while the PYR domain has a conserved catalytic glutamic acid [25]. Multiple sequence alignment of AniP with other well-characterized ThDP enzymes identified the typical 185GDG27N motif and the conserved catalytic glutamic acid E338 (Fig. 2). Based on in vivo fermentation data and bioinformatics analysis, we propose that AniP might catalyze the transfer of a C2 ketol from 3 (donor substrate) to G3P (acceptor substrate), yielding the key intermediate 4 with structural elements for benzylpyrrolidine scaffold assembly (Fig. 1A).Fig. 2 Multiple sequence alignment of siAniP and AniP with E. coli TK (PDB: 2R8P), yeast TK (PDB: 1NGS) and E. coli DXPS (PDB: 2O1S). The conserved residues for ThDP binding are marked with red stars, and the essential residue E338 for catalysis is marked with a black star.

Fig. 2

3.2 An AniP homologue catalyzes condensation reaction between 3 and D-GA not G3P

The previous in vivo feeding experiments with 1,3–13C2-labeled glycerol suggested the role of AniP in bringing together a glycolysis intermediate with 3 to form the molecular backbone of 1 [8]. We attempted to present direct biochemical evidence, but we were unsuccessful in obtaining soluble protein even with the inclusion of commonly used solubilizing fusion tags such as His, GST, Trx, Sumo, MBP, and NusA. The homologous protein siAniP (UniProt ID: A0A918UWT2) from S. inusitatus exhibits 82.5 % sequence identity to AniP. The gene for siAniP clustered with other genes encoding homologous proteins of AniQ, AniN, and AniO, respectively (Fig. 1B). These proteins were essential for the formation of the benzylpyrrolidine scaffold during 1 biosynthesis. We complemented the ΔaniP mutant by expressing sianiP under the control of permE* (Fig. 1C), which completely restored the production of 1. This data proved the equal role of siAniP to that of AniP.

To confirm the function of siAniP, we expressed siAniP in E. coli BL21(DE3) and purified it to near homogeneity using Ni-NTA affinity chromatography. The purity and size (66.8 kDa) of the resulting protein were determined using SDS-PAGE (Fig. S3A). Many ThDP-dependent enzymes behave as active dimers in solution [26], Ni-NTA-purified siAniP was further purified using size-exclusion chromatography (SEC) to identify the active form of siAniP (Fig. S3B). siAniP showed a peak with a retention time earlier than that of the standard bovine serum albumin (BSA, 66 kDa) and close to that of aldolase (158 kDa), but later than catalase (232 kDa) (Fig. S3B), suggesting that siAniP exists as a dimer in solution. In Streptomyces, glycerol can be converted to G3P through a glycerol utilization pathway, and G3P is commonly utilized as an acceptor by other TKs [8]. For the biochemical demonstration of transketolase-type condensation, we first conducted an in vitro enzymatic reaction using 3 as the donor and the commonly used acceptor D/L-G3P in the presence of MgCl2 and ThDP. However, no peak corresponding to 4 could be detected in the reaction system (Fig. 3A). For many TKs and DXPSs, they exhibited low catalytic efficiency on non-phosphorylated substrates, besides the strong preference toward phosphorylated substrates [[27], [28], [29]]. Referring to that, D-GA was then used as the acceptor substrate in the in vitro reactions. A new peak was detected, and the HR-MS analysis revealed that it shared the same retention time and m/z value (249.0738, [M+Na]+, cal 249.0739) as standard 4 (m/z 249.0738, [M+Na]+, cal 249.0739) (Fig. S4). This result proves that siAniP is a TK-like enzyme involved in 1 biosynthesis responsible for transferring a benzene-substituted C2 ketol from donor 3 to the acceptor D-GA, instead of the typical G3P. No DXPSs and TKs have been reported to utilize aromatic keto acids as donor substrates. This catalytic activity of siAniP introduces a new type of reaction.Fig. 3 In vitro activity assay of siAniP. (A) HPLC profile of the reactions catalyzed by siAniP with substrate 3, D-GA or G3P in the presence of ThDP and Mg2+ (λ = 280 nm). The boiled inactive siAniP incubated with 3 and D-GA (III) is used as a negative control. (I) and (II) are 4 and 3 standards. (IV) HPLC profile of siAniP incubated with 3 and G3P. (V) HPLC profile of siAniP incubated with 3 and D-GA. (VI) HPLC profile of siAniP incubated with 3 as the sole substrate. (B) Schematic reactions catalyzed by siAniP.

Fig. 3

Additionally, when 3 was used as the unique substrate, another new compound 5 with a m/z of 271.0991 ([M − H]-) was also observed. For structural elucidation, we purified it from large-scale reaction systems. The NMR analysis (Table S4) confirmed that 5 was an aromatic acyloin, resulting from intermolecular symmetrical coupling of 3 (Fig. 3B). Moreover, 5 could also be detected in the reaction system composed of 3 and GA or G3P (Fig. 3B). This intermolecular symmetrical coupling is similar to the intermolecular coupling of pyruvate producing acetolactate, catalyzed by ALS [30] and DXPS [29]. No TK has been reported to catalyze reactions with aromatic keto acids as substrates. No activity was observed for the EDTA-treated siAniP in the absence of Mg2+ and ThDP (Fig. S5). The condensation activity of siAniP can be restored upon their addition (Fig. S5). All this data proves that siAniP is strictly dependent on Mg2+ and ThDP. To the best of our knowledge, siAniP is the first TK enzyme that catalyzes the transfer of a C2 ketol and symmetrical intermolecular coupling using aromatic keto acids as donor substrates.

3.3 Amino acid residues are essential for substrate binding in siAniP

The unique characteristics of siAniP, such as its activity on phenylpyruvic acids and preference for non-phosphorylated substrates GA, distinguish it as a transketolase that differs from other known TKs and DXPSs. We attempted to determine the crystal structure to understand the catalytic characteristics but we were unable to obtain satisfactory diffraction data for X-ray analysis. Alternatively, we generated a predicted homodimer model of siAniP using AlphaFold 2 [31]. The top-ranked model had pTM and pLDDT scores of 0.920 and 0.98, indicating confident prediction. Furthermore, a DALI search was conducted to identify structural homologues of siAniP in the PDB database. Despite the low sequence similarity, the overall structure of siAniP shows similarity to several DXPSs and TKs, with a root mean square distance (RMSD) of approximately 2.5 Å and 10 Å for equivalent Cα atoms, respectively (Table S5). This finding was consistent with the evolutionary analysis (Fig. S2). Similar to TK and DXPS, each monomer of siAniP consists of three unique domains (Fig. S6A). Domain I, known as the PP domain (residues 1–290), and Domain II, the Pyr domain (residues 290–454), are essential for the formation of the active site and the binding of ThDP. Domain III (residues 455–607) plays a crucial role in establishing extensive interactions at the dimer interface (Fig. S6A). Structural alignment of siAniP with E. coli TK (PDB: 2R8P) and E. coli DXPS (PDB: 2O1S) reveals that the active sites are highly conserved (Figs. S6B and S6C). Although siAniP shares a similar active site organization and ThDP binding mode with TK, the conserved amino acid residues responsible for substrate recognition in TK and DXPS are absent in siAniP. 2-p-hydroxyphenylacetyl-ThDP, the adduct of 3 and ThDP was docked into the active site of siAniP using CB-Dock2 (Score: 5.6) (Fig. 4A). In the model, ThDP is bound in a classical V conformation and can be well accommodated without steric hindrance in the active site. Specifically, the pyrophosphate moiety of ThDP was bound to the divalent metal ion, which was coordinated by the motif 185GDG27N. The thiazole ring of ThDP is sandwiched by the side chains of Y362 and L336 through π-π stacking and van der Waals interactions. The side chain of E338 is close to N1 of aminopyrimidine and may facilitate the deprotonation of the C2 of thiazolium, which is crucial for the catalysis. In the substrate tunnel formed by I220, H275, R322 and W391, the benzene ring of the substrate aligns approximately 90° with the thiazole ring of ThDP. W391 is spatially corresponding to D469 of E. coli TK; I220, positioned opposite to W391, potentially introduces van der Waals contacts with the benzene ring of the substrate. The side chain of R322 contacts the hydroxyl group of the substrate through a hydrogen bond (3.3 Å). H275, corresponding to H261 of E. coli TK, forms an approximately 90° angle with the substrate benzene ring and might form a hydrogen bond with the hydroxyl group of the substrate (Fig. 4A).Fig. 4 Structural analysis of the substrate-binding site and a mutagenesis study of siAniP. (A) Molecular docking of siAniP with 2-p-hydroxyphenylacetyl ThDP intermediate (yellow). The residues I220, H275, R322, and W391, which interact with the benzene ring of the substrate, are shown in green. (B) The relative activity of siAniP mutants.

Fig. 4

To investigate the role of specific residues (I220, H275, R322 and W391), we conducted site-directed mutagenesis. All the recombinant siAniP mutants could be successfully expressed and purified from E. coli (Fig. S3A). We then performed the enzymatic reaction using 3 as substrate, and compared the yield of product 5. Referring to the wild-type siAniP, the activity of all the mutants was dramatically reduced, especially the mutants R322D and W391D, which showed a complete loss of activity (Fig. 4B). R322A lost the hydrogen bond interaction with the substrate and only exhibited 12 % activity, while R322D completely lost its activity due to electrostatic repulsion with the substrate. W391 may contribute to the binding of the substrate through π-π stacking. The replacement with aspartic acid (W391D) abolished its activity, while the alanine mutation (W391A) may disrupt the interaction (Fig. 4B and Fig. S7). Conversely, replacement with aromatic phenylalanine (W391F) and tyrosine (W391Y) retained activity to a certain degree due to the shared benzyl ring. I220N retained 82 % of its activity, while I220R and I220D only possessed 24 % and 5 % activity, respectively. These findings suggest a nonpolar interaction between the side chain of I220 and the benzene ring of substrate. This interaction could be decreased by a mutation with an acidic/basic amino acid, likely due to electrostatic repulsion. H275 possesses both positively charged and aromatic ring side chain. The positively charged H275 can establish hydrogen bonds or electrostatic interactions with the substrate, while its aromatic ring side chain participates in π-π conjugation with the benzene ring of the substrate. Although the H275 side chain is orthogonal to the substrate benzene ring in this model, it may interact with the benzene ring of the second substrate. H275D and H275N exhibited approximately 30 % activity (Fig. 4B and Fig. S7). These observations have validated the significance of the identified residues and suggested their involvement in substrates binding.

3.4 siAniP exhibited high specificity for aromatic keto acids and promiscuity towards aldehydes

Previous crystallographic studies have shown that 1 binding to eukaryotic ribosomes (Leishmanial major) by inserting its para-methoxy phenyl group into the hydrophobic groove of the ribosome's A site, and thus prevents the binding of aminoacyl-tRNA and interrupts peptide chain elongation [32]. Modification of the methoxyphenyl group of 1 with alternative ethers leads to analogs with enhanced anti-parasitic efficacy and selectivity [33]. These findings indicate that the para-phenyl moiety provides an effective location for introducing substituents that confer higher biological activities. To that end, we tested the ability of siAniP to act on various phenylpyruvic acid analogs. We selected five commercially available analogs, including three analogs with different substituents at the para position: phenylpyruvic acid (D2), p-nitrophenylpyruvic acid (D3), p-fluorophenylpyruvic acid (D4), and two analogs with different aromatic rings: indole-3-pyruvic acid (D5), 3-(2-naphthyl) pyruvic acid (D6) (Fig. 5A). In the in vitro reaction system using D-GA (A1) as an acceptor, D3, D5, and D6 could be transformed into the corresponding products with conversion rates of 39.9 %, 44.1 % and 63.7 %, respectively. However, D2 and D4 could not be converted by siAniP (Fig. 5B).Fig. 5 Substrate promiscuity of siAniP. (A) Structure of substrates used to assess the catalytic activity of siAniP. (B) Conversion rate of siAniP towards different keto acids and aldehyde combinations.

Fig. 5

ThDP-dependent enzymes are versatile biocatalysts for the synthesis of α-hydroxy ketones, which are an important class of organic compounds with widespread applications in pharmacy, chemistry, and industry [34,35]. To fully exploit the potential of siAniP in synthesizing α-hydroxy ketones, we further conducted a combinatorial test using the donors and acceptors mentioned above. Moreover, we supplemented the donor substrate with aliphatic keto acids β-hydroxypyruvic acid (D7) and pyruvic acid (D8), which have been reported to be recognized as donor substrates by TK and DXPS, respectively [36]. For the aldehyde acceptor substrates, we collected two aliphatic aldehydes (A1, A2) and three aromatic aldehydes (A3, A4, A5) (Fig. 5A). Firstly, each donor substrate was mixed with five aldehyde acceptors, resulting in the production of 17 different α-hydroxy ketones (Fig. S8, S9 and S10). Among them, the conversion rates of D2A3, D2A5 and D3A4 were comparable to that of D1A1. More D3A1, D5A1, D6A1, D1A3, D6A3 and D6A4 could be produced. siAniP showed a preference for D2 and A4, which had the highest conversion rate (Fig. 5B). Given the intermolecular condensation between two molecules of 3 by siAniP (Fig. 3), each donor substrate was then used individually or mixed with seven other keto acids. Seven α-hydroxy ketone products were detected. Two of them (D3D3, D6D6) were formed through the symmetric coupling of two identical keto acids, while the other five products (D1D3, D1D6, D3D4, D3D6 and D4D6) were produced through the asymmetric condensation of two distinct keto acid substrates (Figs. S8 and S9). As can be seen from these results, all aldehydes and phenylpyruvic acid analogs used here can be catalyzed by siAniP, except for aliphatic keto acids D7 and D8. Taken together, siAniP exhibited high specificity for aromatic keto acids and promiscuity towards aldehydes.

3.5 AniP-like proteins are mainly distributed in candidate BGCs encoding benzylpyrrolidines

The above characterization of siAniP identified it as a transketolase with a combination of features of both TK and ALS. To explore the distribution of siAniP-like TK, AniP was used as a probe to retrieve its homologues via UniProt. The results showed that there were at least 10,000 homologues (>30 % identity) in various organisms, and most sequences were approximately 300 amino acids (AA) in length. Further analysis indicated that the homologous proteins of siAniP-like TK are widely present in bacteria, such as Actinomycetota (681), Bacillota (2919), Pseudomonadota (1392), and Bacteroidota (1684). 415 homologous proteins originate from Eukaryota, and 345 are derived from Archaea. Small numbers of these homologues (of 600 AA) are primarily found in Streptomyces, Chromobacterium and Pseudomonas. The general distribution suggests that siAniP-like TK may be a large conserved family.

During 1 biosynthesis, aniN, aniP and aniQ were proved to be essential for benzylpyrrolidine assembly. The versatile catalytic ability of siAniP towards phenylpyruvic acid analogs, combined with the reported tolerance of the other two enzymes, presents a potential strategy for the structural modification of benzylpyrrolidine. Perhaps other siAniP homologues with different preferences toward substrates would further extend the structural diversity of 1. We then performed a sequence similarity network (SSN)-based genome neighborhood network (GNN) analysis to investigate genes clustered with AniP, as well as their distribution across different microbes. The SSN was generated for AniP homologues in the UniProt database and ﬁltered to include edges with an alignment score threshold of 61 (SSN61, ∼30 % sequence identity) and proteins with a minimum length of 500 residues. We observed that the 238 sequences are primarily grouped into two distinct clusters (Fig. 6A). Although most nodes are located in Cluster 1, further BLAST analysis revealed that these nodes only exhibit ∼30 % sequence similarity with AniP at their N-terminal (∼300 residues). AniP and siAniP are both located in Cluster 2, and the nodes within Cluster 2 share sequence similarity with AniP covering the full length (>500 AA). Most nodes in Cluster 2 originate from Streptomyces (23), 7 are derived from Chromobacterium, and 5 are derived from Pseudomonas. Meanwhile, all AniP homologues in Cluster 2 are surrounded by AniQ (aminotransferase) and AniN (short-chain dehydrogenase) homologues. While the tailoring enzymes for 1 biosynthesis were not always conserved in these clusters: (i) the AniO (glycosyltransferase) homologue was absent in all Chromobacterium and Pseudomonas-sourced gene clusters, (ii) several Streptomyces and Pseudomonas-sourced gene clusters do not contain an AniI (acyltransferase) homologue, (iii) all Streptomyces-derived gene clusters without AniI contain an oxidoreductase and an aldo/keto reductase (Fig. 6B and S11). The diverse arrangement and distribution of AniP and related proteins involved in 1 biosynthesis suggest untapped possibilities for benzylpyrrolidine analogs biosynthesis, and the homologous gene clusters may serve as a reservoir for future biocatalyst development.Fig. 6 Bioinformatics analysis of siAniP and its homologues. (A) Sequence similarity network (SSN) of AniP. Both AniP and siAniP were located in Cluster2. (B) comparative analysis of BGCs picked from GNNs of AniP-like proteins using clinker. AniQ, AniP, and AniN were conserved in all of the selected BGCs.

Fig. 6

4 Discussion

1 was initially demonstrated to selectively inhibit protein translation in eukaryotes, but it has been shown to target various organisms through multiple mechanisms of action, making it a potential multiple function drug [36]. It inhibits translation by binding to the 60S ribosomal subunit in the 80S ribosome system of eukaryotic cells [2,7,8]. The 2397 (2055) rRNA nucleotide has been suggested to contribute to the selective activity of 1 against eukaryotic organisms. In bacteria, this site is a conserved cytosine, but in most eukaryotes, it is an adenine [33]. Upon binding to the ribosome, the para-phenol moiety of 1 inserts completely into the hydrophobic crevice of the A-site and blocks the access of the incoming aminoacyl-tRNAs, causing the disruption of peptide bond formation [10]. Therefore, the para-methoxyphenyl moiety serves as a starting point for developing new analogs with potent antifungal and insecticidal properties. However, the activation and condensation mechanism of phenylpyruvic acid has not yet been elucidated.

Previous in vivo genetic deletion of aniP and isotope labeled feeding experiment indicated its involvement in the transketolase-type condensation [8]. AniP is a ThDP-dependent enzyme and belongs to the TK subgroup of proteins. TK catalyzes the condensation of phosphoketose and phosphoaldose substrates, and is also involved in the biosynthesis of nucleic acids and aromatic amino acids [26]. TKs have been reported to be involved in the biosynthesis of ecteinascidin 743 [37], rifamycin L [38] and herbicidal phosphonate [39,40]. TK from E. coli has been developed to synthesize an aminodiol intermediate of florfenicol [41]. To date, TKs have played crucial roles in catalyzing the formation of asymmetric carbon-carbon bonds. Further characterization and mechanistic elucidation of TK can enhance the understanding of substrates scope and reaction types, leading to the development of new biocatalysts for synthesizing unnatural precursor molecules in chemistry and pharmaceuticals. However, no TK has been reported to catalyze the condensation of phenylpyruvic and aldehyde.

In this study, the complementation of the aniP gene into the ΔaniP mutant successfully restored the production of 1, supporting its essential role (Fig. 1C). To verify the condensation activity, transketolase siAniP (AniP homologue) was used instead, and it was found to catalyze the condensation between 3 and GA, not G3P (Fig. 3A). This reaction catalyzed by siAniP is distinguished from common reactions between ketoses and hydroxypyruvate catalyzed by typical TKs. Meanwhile, siAniP catalyzed the asymmetric condensation of two different aromatic keto acid substrates (Fig. 3, Fig. 5). For TK-like enzymes, only DXPS has been proved to catalyze the coupling between two molecules of pyruvate to form acetolactate, albeit with lower catalytic efficiency. The catalytic efficiency (kcat/Km) of siAniP on compound 3 was 7.06 mM−1 min−1. In terms of comparison with other TKs, the kcat/Km value of siAniP was 117 times that of TK from Geobacillus stearothermophilus toward pyruvate ((kcat/Km = 0.06 mM−1 min−1) [28]. However, it was much smaller than that of DXPS from E. coli on pyruvate (kcat/Km = 223 mM−1 min−1) [29]. However, siAniP showed specificity towards aromatic keto acids and promiscuity towards aldehydes (Fig. 5). We attempted to investigate the preference mechanism using predicted structures of siAniP. In siAniP, the active sites are located at the interface of domains I and II within a single monomer, resembling those of DXPS but different from TK. In TK, the active site is situated at the dimer interface, composed of domain I from one monomer and domain II from another. The conserved motif 185GDG27N for Mg2+ ion binding and ThDP anchoring was identified (Fig. 2). Based on the docking model of siAniP (Fig. 4A), key residues involved in substrate binding vary significantly. In E. coli TK, the phosphate group of the substrate interacts with the side chains of the conserved residues R358, S385, H461 and R520, which are located at the entrance tunnel of the active site. These residues are conserved in TK and DXPS, but they are not conserved in siAniP (Fig. S12). On the contrary, the residue essential for phosphate group binding, R520 in E. coli TKs (equivalent to R478 in E. coli DXPS), is replaced by D439 in siAniP. The electrostatic repulsion between D439 and the phosphate group of the G3P could block the entry of the substrate into the active site. This may be the reason for the inactivity of siAniP toward G3P (Fig. 3). In the crystal structure of E. coli TK in complex with d-fructose-6-phosphate, it was revealed that the side chains of H26, H100, H261, D469, and H473 form an array of hydrogen bonds with the hydroxyl groups of the sugar. Multiple sequence alignment showed that these residues are highly conserved in yeast TK and human TK (Fig. S12). D469 appears to be the outlier in this histidine-rich motif; however, it plays an indispensable role. Based on the mutation of yeast TK, D469 contributes to substrate binding, selects the correct stereoisomer, and is essential for efficient catalysis [42]. Moreover, D469 has been an important engineering point for expanding substrate specificity and altering product stereochemistry [43,44]. However, the histidine-rich motif in TK is absent in siAniP (Fig. S12). The disparity in substrates, with non-phosphorylated for siAniP and phosphorylated ketose for TK, may explain the absence of an extensive hydrogen bond network for substrate binding, suggesting its non-essential nature. Meanwhile, in the docking model of siAniP with 2-p-hydroxyphenylacetyl-ThDP, four amino acid residues were identified that might possibly contribute to substrate binding (Fig. 4A). The site-directed mutations of those amino acid residues (Fig. 4B) proved their vital role in providing steric interactions within the active site. This might explain its preference for non-phosphorylated substrates.

Considering the structural differences among D1-D4, the molecular size of substituents at the para position appears to significantly influence their recognition or catalytic activity. The inability of siAniP on D2 demonstrated the importance of the substituent on the phenyl group for the interaction between the substrate and the protein. Furthermore, this interaction is not affected by the electronegativity of the substituents, as evidenced by the effective activity of siAniP on D3 with a strong electron-withdrawing group. On the contrary, siAniP cannot utilize D4 with an electron-donating fluorine equivalent to the hydroxyl group in its natural substrate D1. Additionally, the successful condensation activity on D5 and D6 indicated a relatively large substrate-binding pocket of siAniP, suggesting its ability to accommodate substrates with larger phenylpyruvic acid derivatives. All these observations provide clues for future selective modification of 1 with various natural characteristic properties.

Homologous proteins of siAniP can be found in other candidate anisomycin BGCs (Fig. 6B). The BGCs varied in gene composition and gene arrangement (Fig. 6B and Fig. S11). Future characterization of those AniP homologues will provide a potentially potent biocatalyst source for the structural modification of benzylpyrrolidine, working in conjunction with two other essential proteins (AniN and AniQ). In summary, this study aims to expand the catalytic capabilities of TKs and develop an efficient, environmentally friendly biocatalyst for the structural modification of anisomycin through a synthetic biology approach, which is crucial for future research endeavors.

5 Conclusion

In summary, the transketolase AniP homologue, siAniP was able to catalyze the condensation between an aromatic keto acid and an aldehyde, indicating its potential as a potent biocatalyst for α-hydroxy ketone production. siAniP exhibited high specificity for aromatic keto acids, making it an unprecedented TK reported up to date. Based on structural docking analysis and site-directed mutagenesis, we identified conserved motifs and important amino acid residues in siAniP for ThDP and substrate binding. Although there have been studies on TKs involved in natural products biosynthesis, the present study is, to the best of our knowledge, the first TK to catalyze the condensation of aromatic keto acids.

Funding

This work was supported by a grant from the National Key research and development Program of China (2021YFA0909500 ，2021 YFC2100100 , 2021YFC2100600 ), and 10.13039/501100001809 National Natural Science Foundation of China (32170077 , 32170075 ).

CRediT authorship contribution statement

Yongjian Qiao: Investigation, Methodology, Writing – original draft. Junbo Wang: Formal analysis. Dashan Zhang: Formal analysis. Xiaoqing Zheng: Investigation. Baixin Lin: Investigation. Yongkang Huang: Investigation. Yulin Liao: Investigation. Zixin Deng: Resources. Lingxin Kong: Conceptualization, Funding acquisition, Supervision, Writing – review & editing. Delin You: Conceptualization, Funding acquisition, Supervision, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for [this journal (Journal Name)] and was not involved in the editorial review or the decision to publish this article.

Zixin Deng is Founding Editor for Synthetic and Systems Biotechnology. And he was not involved in the editorial review or the decision to publish this article. All authors declare that there are no competing interests.

Appendix A Supplementary datag

The following is the supplementary data to this article:Multimedia component 1

Multimedia component 1

Peer review under responsibility of KeAi Communications Co., Ltd.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.synbio.2024.08.006.
==== Refs
References

1 Moore S.J. Lai H.-E. Li J. Freemont P.S. Streptomyces cell-free systems for natural product discovery and engineering Nat Prod Rep 40 2 2023 228 236 10.1039/D2NP00057A 36341536
2 Abdel-Razek A.S. El-Naggar M.E. Allam A. Morsy O.M. Othman S.I. Microbial natural products in drug discovery Processes 8 4 2020 470 10.3390/pr8040470
3 Poyraz S. Döndaş H.A. Döndaş N.Y. Sansano J.M. Recent insights about pyrrolidine core skeletons in pharmacology Front Pharmacol 14 2023 1239658 10.3389/fphar.2023.1239658
4 Green A.J. Gelfand J.M. Cree B.A. Bevan C. Boscardin W.J. Mei F. Inman J. Arnow S. Devereux M. Abounasr A. Nobuta H. Zhu A. Friessen M. Gerona R. von Büdingen H.C. Henry R.G. Hauser S.L. Chan J.R. Clemastine fumarate as a remyelinating therapy for multiple sclerosis (ReBUILD): a randomised, controlled, double-blind, crossover trial Lancet 390 10111 2017 2481 2489 10.1016/S0140-6736(17)32346-2 29029896
5 Heo Y.-A. Budesonide/glycopyrronium/formoterol: a review in COPD Drugs 81 12 2021 1411 1422 10.1007/s40265-021-01562-6 34342835
6 Vatansever E.C. Yang K.S. Drelich A.K. Kratch K.C. Cho C.-C. Kempaiah K.R. Hsu J.C. Mellott D.M. Xu S. Tseng C.-T.K. Liu W.R. Bepridil is potent against SARS-CoV-2 in vitro Proc Natl Acad Sci USA 118 10 2021 e2012201118 10.1073/pnas.2012201118
7 Tiz B.D. Bagnoli L. Rosati O. Marini F. Santi C. Sancineto L. FDA-approved small molecules in 2022: clinical uses and their synthesis Pharmaceutics 14 11 2022 2538 10.3390/pharmaceutics14112538 36432728
8 Zheng X. Cheng Q. Yao F. Wang X. Kong L. Cao B. Xu M. Lin S. Deng Z. Chooi Y.-H. You D. Biosynthesis of the pyrrolidine protein synthesis inhibitor anisomycin involves novel gene ensemble and cryptic biosynthetic steps Proc Natl Acad Sci USA 114 16 2017 4135 4140 10.1073/pnas.1701361114 28373542
9 Sobin B.A. Tanner F.W. Anisomycin, a new anti-protozoan antibiotic J Am Chem Soc 76 1954 4053
10 Loubresse N.G. Prokhorova I. Holtkamp W. Rodnina M.V. Yusupova G. Yusupov M. Structural basis for the inhibition of the eukaryotic ribosome Nature 513 7519 2014 517 522 10.1038/nature13737 25209664
11 Chen W. Yang W. Zhang C. Liu T. Zhu J. Wang H. Li T. Jin A. Ding L. Xian J. Tian T. Pan B. Guo W. Wang B. Modulation of the p38 MAPK pathway by anisomycin promotes ferroptosis of Hepatocellular Carcinoma through phosphorylation of H3S10 Oxid Med Cell Longev 2022 2022 6986445 10.1155/2022/6986445
12 Macias-Silva M. Vázquez-Victorio G. Hernández-Damián J. Anisomycin is a multifunctional drug: more than just a tool to inhibit protein synthesis Curr Chem Biol 4 2010 124 132 10.2174/187231310791170793
13 Nader K. Schafe G.E. Le Doux J.E. Fear memories require protein synthesis in the amygdala for reconsolidation after retrieval Nature 406 6797 2000 722 726 10.1038/35021052 10963596
14 Quintana V.M. Selisko B. Brunetti J.E. Eydoux C. Guillemot J.C. Canard B. Damonte E.B. Julander J.G. Castilla V. Antiviral activity of the natural alkaloid anisomycin against dengue and Zika viruses Antivir Res 176 2020 104749 10.1016/j.antiviral.2020.104749
15 Wang Q. Kong L. Zheng X. Shen J. Wang J. Zhang D. Qiao Y. Wang J. Deng Z. You D. Acyltransferase AniI, a tailoring enzyme with broad substrate tolerance for high-level production of anisomycin Appl Environ Microbiol 87 14 2021 e00172 10.1128/AEM.00172-21 -21
16 Kong L. Liu J. Zheng X. Deng Z. You D. CtcS, a MarR family regulator, regulates chlortetracycline biosynthesis BMC Microbiol 19 1 2019 279 10.1186/s12866-019-1670-9 31823730
17 Sievers F. Wilm A. Dineen D. Gibson T.J. Karplus K. Li W. Lopez R. McWilliam H. Remmert M. Söding J. Thompson J.D. Higgins D.G. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega Mol Syst Biol 7 2011 539 10.1038/msb.2011.75 21988835
18 Robert X. Gouet P. Deciphering key features in protein structures with the new ENDscript server Nucleic Acids Res 42 2014 W320 W324 10.1093/nar/gku316 24753421
19 Tamura K. Stecher G. Kumar S. MEGA11: molecular evolutionary genetics analysis version 11 Mol Biol Evol 38 7 2021 3022 3027 10.1093/molbev/msab120 33892491
20 Xie J. Chen Y. Cai G. Cai R. Hu Z. Wang H. Tree Visualization by One Table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees Nucleic Acids Res 51 W1 2023 W587 W592 10.1093/nar/gkad359 37144476
21 Oberg N. Zallot R. Gerlt J.A. EFI-EST, EFI-GNT, and EFI-CGFP: enzyme function initiative (EFI) web resource for genomic enzymology tools J Mol Biol 435 14 2023 168018 10.1016/j.jmb.2023.168018
22 Kochetov G.A. Solovjeva O.N. Structure and functioning mechanism of transketolase Biochim Biophys Acta, Biochimica et Biophysica Acta (BBA) - Proteins Proteom 1844 9 2014 1608 1618 j.bbapap.2014.06.003
23 Johnston M.L. Freel Meyers C.L. Revealing donor substrate-dependent mechanistic control on DXPS, an enzyme in bacterial central metabolism Biochemistry 60 12 2021 929 939 10.1021/acs.biochem.1c00019 33660509
24 Duggleby R.G. Domain relationships in thiamine diphosphate-dependent enzymes Acc Chem Res 39 8 2006 550 557 10.1021/ar068022z 16906751
25 Costelloe S.J. Ward J.M. Dalby P.A. Evolutionary analysis of the TPP-dependent enzyme family J Mol Evol 66 1 2008 36 49 10.1007/s00239-007-9056-2 18043855
26 Liu Z. Xiao C. Lin S. Tittmann K. Dai S. Multifaceted role of the substrate phosphate group in transketolase catalysis ACS Catal 14 1 2024 355 365 10.1021/acscatal.3c04543
27 Abdoul-Zabar J. Sorel I. Hélaine V. Charmantray F. Devamani T. Yi D. de Berardinis V. Louis D. Marlière P. Fessner W.-D. Hecquet L. Thermostable transketolase from Geobacillus stearothermophilus: characterization and catalytic properties Adv Synth Catal 355 1 2013 116 128 10.1002/adsc.201200590
28 Saravanan T. Junker S. Kickstein M. Hein S. Link M.K. Ranglack J. Witt S. Lorillière M. Hecquet L. Fessner W.-D. Donor promiscuity of a thermostable Transketolase by directed evolution: efficient complementation of 1-deoxy-d-xylulose-5-phosphate synthase activity Angew Chem Int Ed Engl 56 19 2017 5358 5362 10.1002/anie.201701169 28378514
29 Brammer L.A. Meyers C.F. Revealing substrate promiscuity of 1-deoxy-D-xylulose 5-phosphate synthase Org Lett 11 20 2009 4748 4751 10.1021/ol901961q 19778006
30 Gedi V. Yoon M.-Y. Bacterial acetohydroxyacid synthase and its inhibitors--a summary of their structure, biological activity and current status FEBS J 279 6 2012 946 963 10.1111/j.1742-4658.2012.08505.x 22284339
31 Jumper J. Evans R. Pritzel A. Green T. Figurnov M. Ronneberger O. Tunyasuvunakool K. Bates R. Žídek A. Potapenko A. Bridgland A. Meyer C. Kohl S.A. Ballard A.J. Cowie A. Romera-Paredes B. Nikolov S. Jain R. Adler J. Back T. Petersen S. Reiman D. Clancy E. Zielinski M. Steinegger M. Pacholska M. Berghammer T. Bodenstein S. Silver D. Vinyals O. Senior A.W. Kavukcuoglu Koray Kohli P. Hassabis D. Highly accurate protein structure prediction with AlphaFold Nature 596 7873 2021 583 589 10.1038/s41586-021-03819-2 34265844
32 Hansen J.L. Moore P.B. Steitz T.A. Structures of five antibiotics bound at the peptidyl transferase center of the large ribosomal subunit J Mol Biol 330 5 2003 1061 1075 S0022-2836(03)00668-5 12860128
33 Nguyen A.M.T. Shalev-Benami M. Rosa-Teijeiro C. Ibarra-Meneses A.V. Yonath A. Bashan A. Jaffe C.L. Olivier M. Fernandez-Prada C. Lubell W.D. Systematic exploration of functional group relevance for anti-leishmanial activity of anisomycin Biomedicines 11 9 2023 2541 10.3390/biomedicines11092541 37760981
34 Giovannini P.P. Bortolini O. Massi A. Thiamine-diphosphate-dependent enzymes as catalytic tools for the asymmetric benzoin-type reaction Eur J Org Chem 47 45 2016 10.1002/ejoc.201600228
35 Hoyos P. Sinisterra J.-V. Molinari F. Alcántara A.R. de María P.D. Biocatalytic strategies for the asymmetric synthesis of alpha-hydroxy ketones Acc Chem Res 43 2 2010 288 299 10.1021/ar900196n 19908854
36 Arbia G. Gadona C. Casajus H. Nauton L. Charmantray F. Hecquet L. Cross-acyloin condensation of aldehydes catalysed by transketolase variants for the synthesis of aliphatic α-hydroxyketones Green Chem 2024 10.1039/D4GC01373E
37 Peng C. Pu J. Song L. Jian X. Tang M. Tang G. Hijacking a hydroxyethyl unit from a central metabolic ketose into a nonribosomal peptide assembly line Proc Natl Acad Sci U S A 109 22 2012 8540 8545 10.1073/pnas.1204232109 22586110
38 Qi F. Lei C. Li F. Zhang X. Wang J. Zhang W. Fan Z. Li W. Tang G. Xiao Y. Zhao G. Li S. Deciphering the late steps of rifamycin biosynthesis Nat Commun 9 1 2018 2342 10.1038/s41467-018-04772-x 29904078
39 Zhu Y. Shiraishi T. Lin J. Inaba K. Ito A. Ogura Y. Nishiyama M. Kuzuyama T. Complete biosynthetic pathway of the phosphonate phosphonothrixin: two distinct thiamine diphosphate-dependent enzymes divide the work to form a C-C bond J Am Chem Soc 144 37 2022 16715 16719 10.1021/jacs.2c06546 36067081
40 Bown L. Hirota R. Goettge M.N. Cui J. Krist D.T. Zhu L. Giurgiu C. van der Donk W.A. Ju K.-S. Metcalf W.W. A novel pathway for biosynthesis of the herbicidal phosphonate natural product phosphonothrixin is widespread in Actinobacteria J Bacteriol 205 5 2023 e00485 10.1128/jb.00485-22 -22
41 Liu Q. Xie X. Tang M. Tao W. Shi T. Zhang Y. Huang T. Zhao Y. Deng Z. Lin S. One-pot asymmetric synthesis of an aminodiol intermediate of florfenicol using engineered transketolase and transaminase ACS Catal 11 12 2021 7477 7488 10.1021/acscatal.1c01229
42 Nilsson U. Meshalkina L. Lindqvist Y. Schneider G. Examination of substrate binding in thiamin diphosphate-dependent transketolase by protein crystallography and site-directed mutagenesis J Biol Chem 272 3 1997 1864 1869 10.1074/jbc.272.3.1864 8999873
43 Hailes H.C. Rother D. Müller M. Westphal R. Ward J.M. Pleiss J. Vogel C. Pohl M. Engineering stereoselectivity of ThDP-dependent enzymes FEBS J 280 24 2013 6374 6394 10.1111/febs.12496 24034356
44 Yi D. Saravanan T. Devamani T. Charmantray F. Hecquet L. Fessner W.-D. A thermostable transketolase evolved for aliphatic aldehyde acceptors Chem Commun 51 3 2015 480 483 10.1039/c4cc08436e
