Cystathionine γ‐lyase of <i>Saccharomyces cerevisiae</i>: Structural gene and cystathionine γ‐synthase activity

Ono, Bun-ichiro; Ishii, Nobuya; Naito, Kazuhide; Miyoshi, Shin‐ichi; Shinoda, Sumió; Yamamoto, Shigeko; Ohmori, Shinji

doi:10.1002/yea.320090409

Cited by 11 publications

(3 citation statements)

References 31 publications

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“…ORF1 encodes a protein of 423 amino acids with a deduced molecular mass of 45 kDa. Comparison of the proteins in databases with the sequences encoded by these two ORFs showed that the product of ORF1 was similar to cystathionine-c-lyase proteins from S. cerevisiae (encoded by CYS3; Ono et al 1992Ono et al , 1993, Caenorhabditis elegans, human (Lu et al 1992) and rat (Erickson et al 1990), showing 58.3%, 41.8%, 43.7% and 43.5% amino acid sequence identity, respectively). Previous determination of the N-terminal amino acid sequence of the puri®ed S. cerevisiae cystathionine-c-lyase proved unequivocally that it corresponds to the protein encoded by the CYS3 gene (Ono et al 1993).…”

Section: Resultsmentioning

confidence: 96%

Characterization of the reverse transsulfuration gene mecB of Acremonium chrysogenum, which encodes a functional cystathionine-γ-lyase

et al. 2001

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In Acremonium chrysogenum, biosynthesis of cysteine for the formation of cephalosporin has been proposed to occur through the reverse transsulfuration pathway. A gene, named mecB, has been cloned from an A. chrysogenum C10 genomic library in lambdaEMBL3-ble. The cloned DNA fragment encodes a protein of 423 amino acids with a deduced molecular mass of 45 kDa that shows great similarity to cystathionine-gamma-lyases from Saccharomyces cerevisiae and other eukaryotic organisms. The protein was shown to be functional because it restores growth on methionine to A. nidulans C47 (mecB10), a mutant that is known to be defective in cystathionine-gamma-lyase. The cloned gene did not complement A. nidulans mecA or metG mutants. Enzyme activity assays confirmed that the cloned mecB gene encodes a cystathionine-gamma-lyase activity. The mecB gene is present in a single copy in the wild-type A. chrysogenum (Brotzu's strain) and also in the A. chrysogenum strain C10, a high cephalosporin producer. The gene is localized on chromosome VIII (5.3 Mb), as shown by hybridization to A. chrysogenum chromosomes resolved by pulsed-field gel electrophoresis. Transcription of the mecB gene gives rise to a major transcript of 1.5 kb and a minor one of 1.7 kb. The transcript levels were not significantly affected by addition of DL-methionine to the culture, indicating that expression of this gene is not regulated by methionine. The availability of this gene provides a very useful tool for understanding the proposed role of cystathionine-gamma-lyase in splitting cystathionine to supply cysteine for cephalosporin biosynthesis.

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Section: Resultsmentioning

confidence: 96%

Characterization of the reverse transsulfuration gene mecB of Acremonium chrysogenum, which encodes a functional cystathionine-γ-lyase

et al. 2001

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“…It is likely that Saccharomyces cerevisiae has a bifunctional CGL/CGS. Though its CGS activity was only assayed using the elimination reaction with O-acetylhomoserine (so cystathionine formation was never directly established) (42), the structures of the binding sites of this yeast enzyme and E. coli CGS are superimposable, suggesting it also may possess CGS activity (6). Streptomyces phaeochromogenes has a single protein shown both to produce cystathionine by a replacement reaction with homoserine and cysteine (43) and to have CGL activity (8).…”

Section: Discussionmentioning

confidence: 99%

Functional Demonstration of Reverse Transsulfuration in the Mycobacterium tuberculosis Complex Reveals That Methionine Is the Preferred Sulfur Source for Pathogenic Mycobacteria

Wheeler

Coldham

Keating

et al. 2005

Journal of Biological Chemistry

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Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of 35 S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine ␥-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine ␥-synthase (CGS), but demonstration of a ␥-elimination reaction with cystathionine as well as the ␥-replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M.tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine.

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“…On the other hand, reverse transsulfuration, catalyzed by the en-zymes cystathionine β-synthase (CBS) and CGL, is known only in fungi and mammals (Flavin, 1971;Nagasawa et al, 1984). yCGL has also been purified from a yeast strain devoid of the OAH/OAS sulfhydrylase enzyme (Ono et al, 1993). CGLs from yeast (Yamagata, 1993) and man (Steegborn et al, 1999) have been cloned, expressed and physiochemically and enzymatically characterized.…”

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confidence: 99%

Determinants of Enzymatic Specificity in the Cys-Met-Metabolism PLP-Dependent Enzyme Family: Crystal Structure of Cystathionine γ-Lyase from Yeast and Intrafamiliar Structure Comparison

Messerschmidt¹,

Worbs²,

Steegborn³

et al. 2003

Biological Chemistry

106

220

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The crystal structure of cystathionine gamma-lyase (CGL) from yeast has been solved by molecular replacement at a resolution of 2.6 A. The molecule consists of 393 amino acid residues and one PLP moiety and is arranged in the crystal as a tetramer with D2 symmetry as in other related enzymes of the Cys-Met-metabolism PLP-dependent family like cystathionine beta-lyase (CBL). A structure comparison with other family members revealed surprising insights into the tuning of enzymatic specificity between the different family members. CGLs from yeast or human are virtually identical at their active sites to cystathionine gamma-synthase (CGS) from E. coli. Both CGLs and bacterial CGSs exhibit gamma-synthase and gamma-lyase activities depending on their position in the metabolic pathway and the available substrates. This group of enzymes has a glutamate (E333 in yeast CGL) which binds to the distal group of cystathionine (CTT) or the amino group of cysteine. Plant CGSs use homoserine phosphate instead of O-succinyl-homoserine as one substrate. This is reflected by a partially different active site structure in plant CGSs. In CGL and CBL the pseudosymmetric substrate must dock at the active site in different orientations, with S in gamma-position (CBL) or in delta-position (CGL). The conserved glutamate steers the substrate as seen in other CGLs. In CBLs this position is occupied by either tyrosine or hydrophobic residues directing binding of CTT such that S is in the in gamma-position. In methionine gamma-lyase a hydrophic patch operates as recognition site for the methyl group of the methionine substrate.

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Cystathionine γ‐lyase of Saccharomyces cerevisiae: Structural gene and cystathionine γ‐synthase activity

Cited by 11 publications

References 31 publications

Characterization of the reverse transsulfuration gene mecB of Acremonium chrysogenum, which encodes a functional cystathionine-γ-lyase

Characterization of the reverse transsulfuration gene mecB of Acremonium chrysogenum, which encodes a functional cystathionine-γ-lyase

Functional Demonstration of Reverse Transsulfuration in the Mycobacterium tuberculosis Complex Reveals That Methionine Is the Preferred Sulfur Source for Pathogenic Mycobacteria

Determinants of Enzymatic Specificity in the Cys-Met-Metabolism PLP-Dependent Enzyme Family: Crystal Structure of Cystathionine γ-Lyase from Yeast and Intrafamiliar Structure Comparison

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