Characterization of (<i>R</i>)-2-Hydroxyisocaproate Dehydrogenase and a Family III Coenzyme A Transferase Involved in Reduction of<scp>l</scp>-Leucine to Isocaproate by<i>Clostridium difficile</i>

Kim, Jihoe; Darley, Daniel J.; Selmer, Thorsten; Buckel, Wolfgañg

doi:10.1128/aem.00772-06

Cited by 62 publications

(60 citation statements)

References 44 publications

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“…In contrast, the expression of the bcd2 operon, involved in acetyl-CoA to butyryl-CoA synthesis, and of genes encoding two phosphate butyryl-transferases (CD0112 and CD0715) and two butyrate kinases (CD0113 and CD2379) increased 5-to 10-fold at the onset of stationary phase. The genes involved in amino acid fermentations were not differentially expressed, with the exception of the ldhA gene and the hadA operon required for L-leucine reduction, which were induced after 10 h of growth (31). Finally, several genes involved in acetyl-CoA production from pyruvate, CO 2 , or CO or related to electron transfer systems, including genes encoding flavodoxins, ferredoxins, flavoproteins, oxidoreductases, ATP synthases, and hydrogenases, were also induced at the onset of stationary phase.…”

Section: Resultsmentioning

confidence: 94%

The Key Sigma Factor of Transition Phase, SigH, Controls Sporulation, Metabolism, and Virulence Factor Expression in Clostridium difficile

et al. 2011

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Toxin synthesis in Clostridium difficile increases as cells enter into stationary phase. We first compared the expression profiles of strain 630E during exponential growth and at the onset of stationary phase and showed that genes involved in sporulation, cellular division, and motility, as well as carbon and amino acid metabolism, were differentially expressed under these conditions. We inactivated the sigH gene, which encodes an alternative sigma factor involved in the transition to post-exponential phase in Bacillus subtilis. Then, we compared the expression profiles of strain 630E and the sigH mutant after 10 h of growth. About 60% of the genes that were differentially expressed between exponential and stationary phases, including genes involved in motility, sporulation, and metabolism, were regulated by SigH, which thus appears to be a key regulator of the transition phase in C. difficile. SigH positively controls several genes required for sporulation. Accordingly, sigH inactivation results in an asporogeneous phenotype. The spo0A and CD2492 genes, encoding the master regulator of sporulation and one of its associated kinases, and the spoIIA operon were transcribed from a SigH-dependent promoter. The expression of tcdA and tcdB, encoding the toxins, and of tcdR, encoding the sigma factor required for toxin production, increased in a sigH mutant. Finally, SigH regulates the expression of genes encoding surface-associated proteins, such as the Cwp66 adhesin, the S-layer precursor, and the flagellum components. Among the 286 genes positively regulated by SigH, about 40 transcriptional units presenting a SigH consensus in their promoter regions are good candidates for direct SigH targets.

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Section: Resultsmentioning

confidence: 94%

The Key Sigma Factor of Transition Phase, SigH, Controls Sporulation, Metabolism, and Virulence Factor Expression in Clostridium difficile

et al. 2011

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“…Members of this family catalyze the reaction via a ternary complex, which renders them insensitive to hydroxylamine or sodium borohydride (21). Results for inhibition experiments with members of CoA-transferase family III applying hydroxylamine and sodium borohydride are ambiguous (20,(55)(56)(57)(58)(59). Hence, both compounds were tested for a potential inactivating effect on Act TBEA6 (see Materials and Methods).…”

Section: Resultsmentioning

confidence: 99%

“…Consequently, this anhydride intermediate should react with sodium borohydride and hydroxylamine, which inactivates the CoAtransferase permanently. Nonetheless, ambiguous results were obtained regarding sensitivity toward these inhibitors (20,(55)(56)(57)(58)(59). Act TBEA6 was only partially inactivated by hydroxylamine and sodium borohydride.…”

Section: Figmentioning

confidence: 99%

Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family

et al. 2013

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“…The kinetic parameters of the purified recombinant Histagged PanE showed that its substrate specificity was very similar to that of the D-manDH2 from Enterococcus and differed from the specificity of known D-HicDHs (see Table S2 in the supplemental material) (34). In particular, like the D-manDHs of Lactobacillus curvatus (29) and Enterococcus faecalis, PanE exhibited a high level of activity toward C-3-branched substrates such as KIV, KMV, and benzoylformate, However, as in HicDHs, a His residue is probably involved in the active site, since activity was inhibited by DEPC treatment.…”

Section: Discussionmentioning

confidence: 99%

The d -2-Hydroxyacid Dehydrogenase Incorrectly Annotated PanE Is the Sole Reduction System for Branched-Chain 2-Keto Acids in Lactococcus lactis

et al. 2009

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Hydroxyacid dehydrogenases of lactic acid bacteria, which catalyze the stereospecific reduction of branchedchain 2-keto acids to 2-hydroxyacids, are of interest in a variety of fields, including cheese flavor formation via amino acid catabolism. In this study, we used both targeted and random mutagenesis to identify the genes responsible for the reduction of 2-keto acids derived from amino acids in Lactococcus lactis. The gene panE, whose inactivation suppressed hydroxyisocaproate dehydrogenase activity, was cloned and overexpressed in Escherichia coli, and the recombinant His-tagged fusion protein was purified and characterized. The gene annotated panE was the sole gene responsible for the reduction of the 2-keto acids derived from leucine, isoleucine, and valine, while ldh, encoding L-lactate dehydrogenase, was responsible for the reduction of the 2-keto acids derived from phenylalanine and methionine. The kinetic parameters of the His-tagged PanE showed the highest catalytic efficiencies with 2-ketoisocaproate, 2-ketomethylvalerate, 2-ketoisovalerate, and benzoylformate (V max /K m ratios of 6,640, 4,180, 3,300, and 2,050 U/mg/mM, respectively), with NADH as the exclusive coenzyme. For the reverse reaction, the enzyme accepted D-2-hydroxyacids but not L-2-hydroxyacids. Although PanE showed the highest degrees of identity to putative NADP-dependent 2-ketopantoate reductases (KPRs), it did not exhibit KPR activity. Sequence homology analysis revealed that, together with the Dmandelate dehydrogenase of Enterococcus faecium and probably other putative KPRs, PanE belongs to a new family of D-2-hydroxyacid dehydrogenases which is unrelated to the well-described D-2-hydroxyisocaproate dehydrogenase family. Its probable physiological role is to regenerate the NAD ؉ necessary to catabolize branched-chain amino acids, leading to the production of ATP and aroma compounds.

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Characterization of (R)-2-Hydroxyisocaproate Dehydrogenase and a Family III Coenzyme A Transferase Involved in Reduction ofl-Leucine to Isocaproate byClostridium difficile

Cited by 62 publications

References 44 publications

The Key Sigma Factor of Transition Phase, SigH, Controls Sporulation, Metabolism, and Virulence Factor Expression in Clostridium difficile

The Key Sigma Factor of Transition Phase, SigH, Controls Sporulation, Metabolism, and Virulence Factor Expression in Clostridium difficile

Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family

The d -2-Hydroxyacid Dehydrogenase Incorrectly Annotated PanE Is the Sole Reduction System for Branched-Chain 2-Keto Acids in Lactococcus lactis

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