Cloning, purification, and characterization of .beta.-cystathionase from E. coli

Dwivedi, Chandra M.; Ragin, Richard C.; Uren, Jack R.

doi:10.1021/bi00256a005

Cited by 75 publications

(57 citation statements)

References 27 publications

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“…The thermosensitive phenotype of the mutant was corrected by pLC4-14, which is known to cover the 65-min region on the E. coli chromosome map (6). The new par mutation is clearly different from either parA or parB, as described in the present report, and will be termed parC.…”

Section: Mg2+ Was Sequestered With Edtamentioning

confidence: 61%

Gene organization in the region containing a new gene involved in chromosome partition in Escherichia coli

et al. 1988

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A new mutation, parC, causing abnormal chromosome segregation was identified in two thermosensitive mutants of Escherichia coli. The thermosensitive growth of the mutants was corrected by pLC4-14 in the Clarke-Carbon collection. This plasmid carries a putative gene which can suppress the ceUl division defect due to ftsl (pbpB) and has hence been termed sufi (sui). The nearness of parC to metC was confirmed, and cotransduction frequency of parC was 59% with metC and 20% with gkc. The parC-sufl region was analyzed by subcloning the chromosome region of pLC4-14. The parC and the sufi gene products were electrophoretically identified as proteins of 75 and 55 kilodaltons (kDa), respectively. The allelism of parC+ on pLC4-14 to parC1215 was confirmed by cloning parCi215. The sufi gene appeared to be dispensable for cell viability, and overproduction of its product caused suppression offtsI. An essential gene coding for a 25-kDa protein was found between the parC and the sufi gene. These three genes were transcribed in the same direction and may be organized into an operon, with parC to the proximal side and with internal promoters at least for the distal genes. The localization of the gene products was examined in maxicells. The sufi protein was synthesized as a precursor which could be chased into a mature form. The major part of the mature form was found in the soluble fraction. The 25-kDa protein was found almost exclusively in the membrane fraction. The parC protein was associated with the membrane fraction in the presence of Mg2`but found in the soluble fraction when Mg2+ was sequestered with EDTA.

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Section: Mg2+ Was Sequestered With Edtamentioning

confidence: 61%

Gene organization in the region containing a new gene involved in chromosome partition in Escherichia coli

et al. 1988

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“…Native gels revealed a faster-migrating CDS activity, which generated sulfide hours after the predominant activity. We suspected that this other protein was MetC, which can degrade cystine as a side reaction (22). The lag before sulfide detection presumably results from the time required to generate cystine from cysteine oxidation.…”

Section: Resultsmentioning

confidence: 99%

Cysteine Catabolism and Cysteine Desulfhydrase (CdsH/STM0458) in Salmonella enterica Serovar Typhimurium

2012

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Cysteine is potentially toxic and can affect diverse functions such as oxidative stress, antibiotic resistance, and swarming motility. The contribution of cysteine catabolism in modulating responses to cysteine has not been examined, in part because the genes have not been identified and mutants lacking these genes have not been isolated or characterized. We identified the gene for a previously described cysteine desulfhydrase, which we designated cdsH (formerly STM0458). We also identified a divergently transcribed gene that regulates cdsH expression, which we designated cutR (formerly ybaO, or STM0459). CdsH appears to be the major cysteine-degrading and sulfide-producing enzyme aerobically but not anaerobically. Mutants with deletions of cdsH and ybaO exhibited increased sensitivity to cysteine toxicity and altered swarming motility but unaltered cysteine-enhanced antibiotic resistance and survival in macrophages. Cysteine desulfhydrase (CDS) degrades cysteine to pyruvate, ammonia, and sulfide (19,26,43). The major CDS in Escherichia coli appears to be tryptophanase (TnaA), which has an apparent K m for cysteine of 11 mM (56). TnaA primarily catabolizes tryptophan, which appears to be abundant in the intestinal tract (29). In E. coli, TnaA can become 10% of soluble protein, which suggests the potential to also degrade cysteine in vivo, despite an unfavorable K m (56). Several factors regulate TnaA expression. TnaA is repressed by glucose, pyruvate, and acetate (6, 9, 10, 31). Expression requires cyclic AMP and is induced by tryptophan, cysteine, indole, growth in alkaline broth, depletion of heme and phosphatidyl glycerol, and anaerobic growth with an alternate electron acceptor (9, 51, 52). From these factors, it could be speculated that TnaA contributes to energy generation by degrading tryptophan and possibly cysteine to pyruvate.Salmonella enterica does not have TnaA, yet cysteine induces a powerful CDS (19,26,43). This CDS has been purified from S. enterica, and similarly to TnaA, it contains pyridoxal-5=-phosphate (42). The k cat /K m ratio for L-cysteine, a measure of catalytic efficiency, is 10,340 mM Ϫ1 s Ϫ1 for S. enterica CDS (calculated from values reported by Kredich et al. [42,43]) and 6.7 mM Ϫ1 s Ϫ1for E. coli TnaA (calculated from values reported by Snell [56]). In other words, the S. enterica CDS appears to be more than three orders of magnitude more efficient than TnaA. To explore the function of the S. enterica CDS and cysteine catabolism, we identified the gene for the major CDS and characterized its regulation and the phenotype of mutants lacking this enzyme. MATERIALS AND METHODSStrains and plasmids. Table 1 lists the strains and plasmids used in this study. All gene deletions and replacements were performed originally into the TT22971 background strain as described previously (21), followed by P22 transduction into the appropriate genetic background, and the products were ensured to be phage free by cross-streaking against P22-H5 on green plate agar (15). The following codons were delet...

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“…As mentioned above, MalY is able to cleave cystathionine to homocysteine, ammonia, and pyruvate in vitro. This reaction, normally catalyzed by the cystathionase encoded by metC (2,13), is an essential step in the biosynthesis of methionine. That is, strains carrying a metC mutation require methionine for growth.…”

Section: Resultsmentioning

confidence: 99%

MalY of Escherichia coli is an enzyme with the activity of a beta C-S lyase (cystathionase)

et al. 1995

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The Escherichia coli maltose system consists of a number of genes whose products are involved in the uptake and metabolism of maltose and maltodextrins. MalT is the central positive gene activator of the regulon and is, together with the cyclic AMP-catabolite gene activator protein system, necessary for the expression of the maltose genes. Expression of malY, a MalT-independent gene, leads to the repression of all MalT-dependent genes. We have purified MalY to homogeneity and found it to be a pyridoxal-5-phosphate-containing enzyme with the enzymatic activity of a ␤C-S lyase (cystathionase). MalY is a monomeric protein of 42,000 to 44,000 Da. Strains expressing MalY constitutively abolish the methionine requirement of metC mutants. The enzymatic activity of MetC, the cleavage of cystathionine to homocysteine, ammonia, and pyruvate, can be catalyzed by MalY. However, the cystathionase activity is not required for the function of MalY in repressing the maltose system. By site-directed mutagenesis, we changed the conserved lysine residue at the pyridoxal phosphate binding site (position 233) of MalY to isoleucine. This abolished ␤C-S lyase activity but not the ability of the protein to repress the maltose system. Also, the overexpression of plasmid-encoded metC did not affect mal gene expression, nor did the deduced amino acid sequence of MetC show homology to that of MalY.The maltose regulon of Escherichia coli consists of two sets of genes which encode proteins involved in the uptake and metabolism of maltose and maltodextrins (␣1,4-linked D-glucose polymers) (29). Expression of the maltose genes is dependent on the presence of the inducer, maltotriose, bound to the positive regulator, MalT (23). Mutants lacking MalT function do not express any mal genes and are phenotypically Mal Ϫ , whereas malT(Con) mutations render mal gene expression independent of (or less dependent on) the inducer, maltotriose (10, 11). The expression of malT, as well as of some of the mal genes, is dependent on activation by the cyclic AMP-catabolite gene activator protein complex (9). Several observations demonstrate that the regulation of the maltose system is not as straightforward as implied above. First, there is the phenomenon of internal induction (12). Second, MalK, the ATP-consuming subunit of the maltose transport system, acts phenotypically as a repressor of the system, affecting the function of MalT (6,18,19,26). The third regulatory circuit affecting mal gene expression is derived from the malI malX malY gene cluster. malI was discovered as a mutation which strongly reduced the high and constitutive expression of a malK-lacZ fusion (14). Molecular analysis of malI (25) and its adjacent genes revealed that malI encodes a typical repressor protein, analogous to LacI and GalR, which prevents the expression of the adjacent and divergently oriented malX malY operon. The gene product of malX is a protein homologous to the enzyme II Glc of the phosphotransferase system. malY encodes a protein with sequence homology, including the ch...

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Cloning, purification, and characterization of .beta.-cystathionase from E. coli

Cited by 75 publications

References 27 publications

Gene organization in the region containing a new gene involved in chromosome partition in Escherichia coli

Gene organization in the region containing a new gene involved in chromosome partition in Escherichia coli

Cysteine Catabolism and Cysteine Desulfhydrase (CdsH/STM0458) in Salmonella enterica Serovar Typhimurium

MalY of Escherichia coli is an enzyme with the activity of a beta C-S lyase (cystathionase)

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