PMID- 10497249
OWN - NLM
STAT- MEDLINE
DCOM- 19991102
LR  - 20190508
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 274
IP  - 40
DP  - 1999 Oct 1
TI  - Selective loss of either the epimerase or kinase activity of
      UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase due to
      site-directed mutagenesis based on sequence alignments.
PG  - 28771-8
AB  - N-Acetylneuraminic acid is the most common naturally occurring sialic acid, as
      well as being the biosynthetic precursor of this group of compounds. UDP-GlcNAc
      2-epimerase/N-acetylmannosamine kinase has been shown to be the key enzyme of
      N-acetylneuraminic acid biosynthesis in rat liver, and it is a regulator of cell 
      surface sialylation. The N-terminal region of this bifunctional enzyme displays
      sequence similarities with prokaryotic UDP-GlcNAc 2-epimerases, whereas the
      sequence of its C-terminal region is similar to sequences of members of the sugar
      kinase superfamily. High level overexpression of active enzyme was established by
      using the baculovirus/Sf9 system. For functional characterization, site-directed 
      mutagenesis was performed on different conserved amino acid residues. The
      histidine mutants H45A, H110A, H132A, H155A, and H157A showed a drastic loss of
      epimerase activity with almost unchanged kinase activity. Conversely, the mutants
      D413N, D413K, and R420M in the putative kinase active site lost their kinase
      activity but retained their epimerase activity. To estimate the structural
      perturbation effect due to site-directed mutagenesis, the oligomeric state of all
      mutants was determined by gel filtration analysis. The mutants D413N, D413K, and 
      R420M as well as H45A were shown to form a hexamer like the wild-type enzyme,
      indicating little influence of mutation on protein folding. Histidine mutants
      H155A and H157A formed mainly trimeric enzyme with small amounts of hexamer.
      Oligomerization of mutants H110A and H132A was also significantly different from 
      that of the wild-type enzyme. Therefore the loss of epimerase activity in mutants
      H110A, H132A, H155A, and H157A can largely be attributed to incorrect protein
      folding. In contrast, the mutation site of mutant H45A seems to be involved
      directly in the epimerization process, and the amino acids Asp-413 and Arg-420 of
      UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase are essential for the
      phosphorylation process. The fact that either epimerase or kinase activity are
      lost selectively provides evidence for the existence of two active sites working 
      quite independently.
FAU - Effertz, K
AU  - Effertz K
AD  - Institut fur Molekularbiologie und Biochemie, Freie Universitat Berlin,
      Arnimallee 22, D-14195 Berlin-Dahlem, Germany. effertzk@zedat.fu-berlin.de
FAU - Hinderlich, S
AU  - Hinderlich S
FAU - Reutter, W
AU  - Reutter W
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Enzyme Inhibitors)
RN  - 0 (Escherichia coli Proteins)
RN  - 4QD397987E (Histidine)
RN  - EC 2.7.1.- (Phosphotransferases (Alcohol Group Acceptor))
RN  - EC 2.7.1.60 (N-acylmannosamine kinase)
RN  - EC 5.1.3.- (Carbohydrate Epimerases)
RN  - EC 5.1.3.14 (UDP acetylglucosamine-2-epimerase)
RN  - EC 5.1.3.14 (wecB protein, E coli)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Base Sequence
MH  - Carbohydrate Epimerases/chemistry/genetics/metabolism
MH  - Catalysis
MH  - Enzyme Inhibitors/pharmacology
MH  - *Escherichia coli Proteins
MH  - Histidine/genetics
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - Phosphotransferases (Alcohol Group Acceptor)/chemistry/genetics/*metabolism
MH  - Rats
MH  - Sequence Homology, Amino Acid
MH  - Spodoptera
EDAT- 1999/09/25 00:00
MHDA- 1999/09/25 00:01
CRDT- 1999/09/25 00:00
PHST- 1999/09/25 00:00 [pubmed]
PHST- 1999/09/25 00:01 [medline]
PHST- 1999/09/25 00:00 [entrez]
AID - 10.1074/jbc.274.40.28771 [doi]
PST - ppublish
SO  - J Biol Chem. 1999 Oct 1;274(40):28771-8. doi: 10.1074/jbc.274.40.28771.