|Gene:||metB||Accession Numbers: EG10582 (MetaCyc), b3939, ECK3931|
Species: Escherichia coli K-12 substr. MG1655
Subunit composition of O-succinylhomoserine(thiol)-lyase / O-succinylhomoserine lyase = [MetB]4
This pyridoxal 5'-phosphate (PLP) dependent enzyme catalyzes a γ-replacement reaction that produces L-cystathionine and succinate from the substrates O-succinylhomoserine and L-cysteine in the transsulfuration pathway for methionine biosynthesis (see pathway methionine biosynthesis I). In the absence of L-cysteine it can catalyze a γ-elimination (deamination) reaction of O-succinylhomoserine to produce 2-oxobutanoate (α-ketobutyrate), ammonia and succinate, although this is considered to be a non-physiological reaction [Holbrook90].
Review: Hondrop, E.R. and R.G. Matthews (2006) "Methionine" EcoSal 126.96.36.199 [ECOSAL].
The enzyme was first purified from Salmonella typhimurium [Guggenheim69]. The native enzyme was later purified from Escherichia coli and characterized as a homotetramer, similar to the Salmonella enzyme [Tran83]. The recombinant enzyme was overproduced in E. coli, purified, and characterized both structurally and kinetically. It contained one PLP binding site per subunit and absorption spectra showed the PLP to be in a Schiff base form with lysine 198 [Holbrook90, Martel87]. The PLP binding sites of purified, native E. coli cystathionine γ-synthase (MetB) and cystathionine β-lyase (MetC) were localized on phosphopyridoxal peptides. The amino acid sequences of these peptides were found to be homologous with the PLP binding site of rat liver cystathionine γ-lyase (γ-cystathionase) [Martel87]. MetB also shares 30% identity and 36% overall amino acid sequence homology with MetC, suggesting a common evolutionary origin [Belfaiza86].
Steady-state kinetic analysis of the enzyme showed a ping-pong mechanism [Holbrook90]. However, later steady state kinetic analysis using continuous assays that employed coupling enzymes suggested that the enzyme has an ordered mechanism at L-cysteine concentrations greater than its Km and a ping-pong mechanism at L-cysteine concentrations lower that its Km [Aitken03]. Spectroscopic analysis techniques showed that the γ-replacement and γ-elimination reactions go through a common intermediate that was identified as an α-imino-β,γ-unsaturated pyridoxamine derivative [Brzovic90].
Site-directed mutagenesis studies probed the role of acidic residues D45 and E325 as determinants of reaction specificity. Substitutions D45F and E325Y interconverted the corresponding residues found in MetC. Although mutations in either position had reduced catalytic efficiency which showed that they are required for substrate binding, complementation experiments suggested that the in vivo reaction specificity was not altered relative to wild-type enzyme. Therefore further work is necessary to define the determinants of reaction specificity [Farsi09].
The crystal structure of the recombinant enzyme has been determined at 1.5 Å resolution [Clausen98].
The apparent molecular mass of the protein complex was determined by gel filtration chromatography [Tran83] or sedimentation equilibrium analysis [Holbrook90] and the apparent molecular mass of the subunit was determined by SDS-PAGE [Tran83, Holbrook90].
|Map Position: [4,126,695 -> 4,127,855]|
Molecular Weight of Polypeptide: 41.55 kD (from nucleotide sequence), 40.0 kD (experimental) [Tran83 ]
Molecular Weight of Multimer: 160.0 kD (experimental) [Tran83]
Unification Links: ASAP:ABE-0012887 , CGSC:515 , DIP:DIP-10192N , EchoBASE:EB0577 , EcoGene:EG10582 , EcoliWiki:b3939 , ModBase:P00935 , OU-Microarray:b3939 , PortEco:metB , PR:PRO_000023209 , Pride:P00935 , Protein Model Portal:P00935 , RefSeq:NP_418374 , RegulonDB:EG10582 , SMR:P00935 , String:511145.b3939 , UniProt:P00935
Relationship Links: InterPro:IN-FAMILY:IPR000277 , InterPro:IN-FAMILY:IPR011821 , InterPro:IN-FAMILY:IPR015421 , InterPro:IN-FAMILY:IPR015422 , InterPro:IN-FAMILY:IPR015424 , Panther:IN-FAMILY:PTHR11808 , PDB:Structure:1CS1 , Pfam:IN-FAMILY:PF01053 , Prosite:IN-FAMILY:PS00868
|Biological Process:||GO:0009086 - methionine biosynthetic process
GO:0008652 - cellular amino acid biosynthetic process [UniProtGOA11a]
|Molecular Function:||GO:0003962 - cystathionine gamma-synthase activity
GO:0030170 - pyridoxal phosphate binding [GOA01a, Holbrook90]
GO:0042802 - identical protein binding [Holbrook90, Tran83]
GO:0003824 - catalytic activity [GOA01a]
GO:0016740 - transferase activity [UniProtGOA11a]
|Cellular Component:||GO:0005737 - cytoplasm
GO:0005829 - cytosol [DiazMejia09]
|MultiFun Terms:||metabolism → biosynthesis of building blocks → amino acids → methionine|
|metabolism → carbon utilization → amino acids|
|metabolism → central intermediary metabolism → threonine catabolism|
Enzymatic reaction of: O-succinylhomoserine(thiol)-lyase
Synonyms: cystathionine γ-synthase, O-succinyl-L-homoserine succinate-lyase (adding cysteine), OSHS
EC Number: 188.8.131.52
The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.
The reaction is favored in the direction shown.
In Pathways: superpathway of S-adenosyl-L-methionine biosynthesis , aspartate superpathway , superpathway of lysine, threonine and methionine biosynthesis I , superpathway of methionine biosynthesis (transsulfuration) , homoserine and methionine biosynthesis , methionine biosynthesis I
In this γ-replacement reaction the succinyl group of O-succinyl-L-homoserine is displaced by the thiol of L-cysteine to produce L-cystathionine [Aitken03]. There was no product inhibition with either L-cystathionine or succinate [Holbrook90].
pH(opt): 7.8 [Holbrook90]
Enzymatic reaction of: O-succinylhomoserine lyase
EC Number: 4.3.1.-
The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.
The reaction is physiologically favored in the direction shown.
This γ-elimination (deamination) reaction occurs in the absence of L-cysteine. O-succinyl-L-homoserine is hydrolyzed to succinate, ammonia and 2-oxobutanoate (α-ketobutyrate) [Holbrook90, Aitken03].
pH(opt): 7.4 [Aitken03]
10/20/97 Gene b3939 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10582.
Aitken03: Aitken SM, Kim DH, Kirsch JF (2003). "Escherichia coli cystathionine gamma-synthase does not obey ping-pong kinetics. Novel continuous assays for the elimination and substitution reactions." Biochemistry 42(38);11297-306. PMID: 14503880
Belfaiza86: Belfaiza J, Parsot C, Martel A, de la Tour CB, Margarita D, Cohen GN, Saint-Girons I (1986). "Evolution in biosynthetic pathways: two enzymes catalyzing consecutive steps in methionine biosynthesis originate from a common ancestor and possess a similar regulatory region." Proc Natl Acad Sci U S A 83(4);867-71. PMID: 3513164
Brzovic90: Brzovic P, Holbrook EL, Greene RC, Dunn MF (1990). "Reaction mechanism of Escherichia coli cystathionine gamma-synthase: direct evidence for a pyridoxamine derivative of vinylglyoxylate as a key intermediate in pyridoxal phosphate dependent gamma-elimination and gamma-replacement reactions." Biochemistry 1990;29(2);442-51. PMID: 2405904
DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114
Duchange83: Duchange N, Zakin MM, Ferrara P, Saint-Girons I, Park I, Tran SV, Py MC, Cohen GN (1983). "Structure of the metJBLF cluster in Escherichia coli K12. Sequence of the metB structural gene and of the 5'- and 3'-flanking regions of the metBL operon." J Biol Chem 1983;258(24);14868-71. PMID: 6361020
Farsi09: Farsi A, Lodha PH, Skanes JE, Los H, Kalidindi N, Aitken SM (2009). "Interconversion of a pair of active-site residues in Escherichia coli cystathionine gamma-synthase, E. coli cystathionine beta-lyase, and Saccharomyces cerevisiae cystathionine gamma-lyase and development of tools for the investigation of their mechanisms and reaction specificity." Biochem Cell Biol 87(2);445-57. PMID: 19370061
Guggenheim69: Guggenheim S, Flavin M (1969). "Cystathionine gamma-synthase from Salmonella. Beta elimination and replacement reactions and inhibition by O-succinylserine." J Biol Chem 244(13);3722-7. PMID: 4893684
Hacham03: Hacham Y, Gophna U, Amir R (2003). "In vivo analysis of various substrates utilized by cystathionine gamma-synthase and O-acetylhomoserine sulfhydrylase in methionine biosynthesis." Mol Biol Evol 20(9);1513-20. PMID: 12832650
Holbrook90: Holbrook EL, Greene RC, Krueger JH (1990). "Purification and properties of cystathionine gamma-synthase from overproducing strains of Escherichia coli." Biochemistry 1990;29(2);435-42. PMID: 2405903
Martel87: Martel A, Bouthier de la Tour C, Le Goffic F (1987). "Pyridoxal 5'phosphate binding site of Escherichia coli beta cystathionase and cystathionine gamma synthase comparison of their sequences." Biochem Biophys Res Commun 1987;147(2);565-71. PMID: 3307782
Tran83: Tran SV, Schaeffer E, Bertrand O, Mariuzza R, Ferrara P (1983). "Appendix. Purification, molecular weight, and NH2-terminal sequence of cystathionine gamma-synthase of Escherichia coli." J Biol Chem 1983;258(24);14872-3. PMID: 6361021
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