PMID- 10529246
OWN - NLM
STAT- MEDLINE
DCOM- 19991124
LR  - 20190613
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 38
IP  - 42
DP  - 1999 Oct 19
TI  - Recombinant human liver betaine-homocysteine S-methyltransferase: identification 
      of three cysteine residues critical for zinc binding.
PG  - 13991-8
AB  - Betaine-homocysteine S-methyltransferase (BHMT; EC 2.1.1.5) catalyzes the 
      transfer of an N-methyl group from betaine to homocysteine to produce 
      dimethylglycine and methionine, respectively. The enzyme is found in the pathway 
      of choline oxidation and is abundantly expressed in liver and kidney. We have 
      recently shown that human BHMT is a zinc metalloenzyme [Millian, N. S., and 
      Garrow, T. A. (1998) Arch. Biochem. Biophys. 356, 93-98]. To facilitate the rapid 
      purification of human BHMT for further physical and mechanistic studies, 
      including characterizing its metal binding properties, we have overexpressed the 
      enzyme in E. coli as a fusion construct which facilitated its subsequent 
      purification by a self-cleavable affinity tag system (IMPACT T7). Using this 
      expression and purification system in conjunction with site-directed mutagenesis, 
      we have identified Cys217, Cys299, and Cys300 as zinc ligands. Mutating any of 
      these Cys residues to Ala results in the complete loss of activity and a 
      significant reduction in the ability of the protein to bind zinc. Comparing the 
      regions of BHMT amino acid sequence surrounding these Cys residues with similar 
      amino acid sequences retrievable from protein databases, we have identified the 
      following motif: G[ILV]NCX(20,100)[ALV]X(2)[ILV]GGCCX(3)PX(2)I, which we propose 
      to be a signature for a family of zinc-dependent methyltransferases that utilize 
      thiols or selenols as methyl acceptors. Some of the members of this family 
      include the vitamin B(12)-dependent methionine synthases, E. coli 
      S-methylmethionine-S-homocysteine methyltransferase, and A. bisulcatus 
      S-methylmethionine-selenocysteine methyltransferase.
FAU - Breksa, A P 3rd
AU  - Breksa AP 3rd
AD  - Department of Food Science and Human Nutrition, University of Illinois, Urbana 
      61801, USA.
FAU - Garrow, T A
AU  - Garrow TA
LA  - eng
GR  - DK52501/DK/NIDDK NIH HHS/United States
GR  - HL58955/HL/NHLBI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Recombinant Fusion Proteins)
RN  - 1398-61-4 (Chitin)
RN  - EC 2.1.1.- (Methyltransferases)
RN  - EC 2.1.1.5 (BHMT protein, human)
RN  - EC 2.1.1.5 (Betaine-Homocysteine S-Methyltransferase)
RN  - EC 5.99.1.3 (DNA Gyrase)
RN  - EC 5.99.1.3 (DNA Topoisomerases, Type II)
RN  - J41CSQ7QDS (Zinc)
RN  - K848JZ4886 (Cysteine)
SB  - IM
MH  - Base Sequence
MH  - Betaine-Homocysteine S-Methyltransferase
MH  - Binding Sites/genetics
MH  - Chitin/genetics
MH  - Cysteine/genetics/*metabolism
MH  - DNA Gyrase
MH  - DNA Topoisomerases, Type II/genetics
MH  - Escherichia coli/enzymology/genetics
MH  - Gene Expression Regulation, Bacterial
MH  - Genetic Vectors/chemical synthesis
MH  - Humans
MH  - Liver/*enzymology
MH  - Methyltransferases/*chemistry/genetics/isolation & purification/*metabolism
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - Recombinant Fusion Proteins/biosynthesis/*chemistry/genetics/*metabolism
MH  - Sequence Alignment
MH  - Software
MH  - Zinc/*metabolism
EDAT- 1999/10/21 09:00
MHDA- 2001/03/28 10:01
CRDT- 1999/10/21 09:00
PHST- 1999/10/21 09:00 [pubmed]
PHST- 2001/03/28 10:01 [medline]
PHST- 1999/10/21 09:00 [entrez]
AID - bi991003v [pii]
AID - 10.1021/bi991003v [doi]
PST - ppublish
SO  - Biochemistry. 1999 Oct 19;38(42):13991-8. doi: 10.1021/bi991003v.