A conserved gene cluster rules anaerobic oxidative degradation of L-ornithine.
ABSTRACT For the ornithine fermentation pathway, described more than 70 years ago, genetic and biochemical information are still incomplete. We present here the experimental identification of the last four missing genes of this metabolic pathway. They encode L-ornithine racemase, (2R,4S)-2,4-diaminopentanoate dehydrogenase, and the two subunits of 2-amino-4-ketopentanoate thiolase. While described only for the Clostridiaceae to date, this pathway is shown to be more widespread.
SourceAvailable from: Denise K Zinniel[Show abstract] [Hide abstract]
ABSTRACT: D-cycloserine is an effective second line antibiotic used as a last resort to treat multi (MDR)- and extensively (XDR)- drug resistant strains of Mycobacterium tuberculosis. D-cycloserine interferes with the formation of peptidoglycan biosynthesis by competitive inhibition of Alanine racemase (Alr) and D-Alanine-D-alanine ligase (Ddl). Although, the two enzymes are known to be inhibited, the in vivo lethal target is still unknown. Our NMR metabolomics work has revealed that Ddl is the primary target of DCS, as cell growth is inhibited when the production of D-alanyl-D-alanine is halted. It is shown that inhibition of Alr may contribute indirectly by lowering the levels of D-alanine thus allowing DCS to outcompete D-alanine for Ddl binding. The NMR data also supports the possibility of a transamination reaction to produce D-alanine from pyruvate and glutamate, thereby bypassing Alr inhibition. Furthermore, the inhibition of peptidoglycan synthesis results in a cascading effect on cellular metabolism as there is a shift toward the catabolic routes to compensate for accumulation of peptidoglycan precursors.Journal of Proteome Research 12/2013; 13(2). DOI:10.1021/pr4010579 · 5.00 Impact Factor
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ABSTRACT: Adenosylcobalamin-dependent ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii utilizes pyridoxal 5'-phosphate to interconvert D-ornithine to 2,4-diaminopentanoate via a multi-step mechanism that involves two hydrogen transfer steps. Herein, we uncover features of the OAM catalytic mechanism that differentiate it from its homologue, the more catalytically promiscuous lysine 5,6-aminomutase. Kinetic isotope effects (KIE) with DL-ornithine-3,3,4,4,5,5-d6 revealed a diminished Dkcat/Km of 2.5 ± 0.4 relative to Dkcat of 7.6 ± 0.5, suggesting slow substrate release from the active site. In contrast, a KIE was not observed on the rate constant associated with Co–C bond homolysis as this step is likely ‘gated’ by the formation of the external aldimine. The role of tyrosine 187, which lies planar to the PLP pyridine ring, was also investigated through site-directed mutagenesis. The 25-fold and 1260-fold reduced kcat for Y187F and Y187A is attributed to a slower rate of external aldimine formation and a diminution in adenosylcobalamin Co–C bond homolysis. Notably, EPR studies of Y187F suggest that the integrity of the active site is maintained as cob(II)alamin and the PLP-organic radical (even at lower concentrations) remain tightly exchanged coupled. Modeling of D-lysine and L-β-lysine into the 5,6-LAM active site reveals fewer and weaker interactions between the substrate and protein compared to OAM. The combined data suggest that the level of protein-substrate interactions in aminomutases not only influences substrate-specificity, but also controlled radical chemistry.Biochemistry 08/2014; 53(33). DOI:10.1021/bi5006706 · 3.19 Impact Factor
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ABSTRACT: 2,4-Diaminopentanoate dehydrogenase (2,4-DAPDH), which is involved in the oxidative ornithine degradation pathway, catalyzes the NAD(+)- or NADP(+)-dependent oxidative deamination of (2R,4S)-2,4-diaminopentanoate (2,4-DAP) to form 2-amino-4-oxopentanoate. A Fervidobacterium nodosum Rt17-B1 gene, Fnod_1646, which codes for a protein with sequence similarity to 2,4-DAPDH discovered in metagenomic DNA, was cloned and overexpressed in Escherichia coli, and the gene product was purified and characterized. The purified protein catalyzed the reduction of NAD(+) and NADP(+) in the presence of 2,4-DAP, indicating that the protein is a 2,4-DAPDH. The optimal pH and temperature were 9.5 and 85°C, respectively, and the half-denaturation time at 90°C was 38 min. Therefore, the 2,4-DAPDH from F. nodosum Rt17-B1 is an NAD(P)(+)-dependent thermophilic-alkaline amino acid dehydrogenase. This is the first thermophilic 2,4-DAPDH reported, and it is expected to be useful for structural and functional analyses of 2,4-DAPDH and for the enzymatic production of chiral amine compounds. Activity of 2,4-DAPDH from F. nodosum Rt17-B1 was suppressed by 2,4-DAP via uncompetitive substrate inhibition. In contrast, the enzyme showed typical Michaelis-Menten kinetics toward 2,5-diaminohexanoate. The enzyme was uncompetitively inhibited by d-ornithine with an apparent Ki value of 0.1 mM. These results suggest a regulatory role for this enzyme in the oxidative ornithine degradation pathway.Journal of Bioscience and Bioengineering 12/2013; DOI:10.1016/j.jbiosc.2013.11.002 · 1.79 Impact Factor