Enzymology and Evolution of the Pyruvate Pathway to 2-Oxobutyrate in Methanocaldococcus jannaschii

Abstract

The archaeon Methanocaldococcus jannaschii uses three different 2-oxoacid elongation pathways, which extend the chain length of precursors in leucine, isoleucine, and coenzyme B biosyntheses. In each of these pathways an aconitase-type hydrolyase catalyzes an hydroxyacid isomerization reaction. The genome sequence of M. jannaschii encodes two homologs of each large and small subunit that forms the hydrolyase, but the genes are not cotranscribed. The genes are more similar to each other than to previously characterized isopropylmalate isomerase or homoaconitase enzyme genes. To identify the functions of these homologs, the four combinations of subunits were heterologously expressed in Escherichia coli, purified, and reconstituted to generate the iron-sulfur center of the holoenzyme. Only the combination of MJ0499 and MJ1277 proteins catalyzed isopropylmalate and citramalate isomerization reactions. This pair also catalyzed hydration half-reactions using citraconate and maleate. Another broad-specificity enzyme, isopropylmalate dehydrogenase (MJ0720), catalyzed the oxidative decarboxylation of ␤-isopropylmalate, ␤-methylmalate, and D-malate. Combined with these results, phylogenetic analysis suggests that the pyruvate pathway to 2-oxobutyrate (an alternative to threonine dehydratase in isoleucine biosynthesis) evolved several times in bacteria and archaea. The enzymes in the isopropylmalate pathway of leucine biosynthesis facilitated the evolution of 2-oxobutyrate biosynthesis through the introduction of a citramalate synthase, either by gene recruitment or gene duplication and functional divergence.

The Metabolic Regulation of Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis Revealed by Transcriptomics and Proteomics

“…If this reaction was drastically inhibited, the energy yield and the processes of the entire TCA cycle as well as the glyoxylate and GABA shunts would have all been significantly affected. Interestingly, some studies confirmed that LeuB (3-isopropylmalate dehydrogenase) is a broad-specificity enzyme that can catalyze the oxidative decarboxylation of 3-methylmalate, as well as D-and L-malate in addition to its cognate substrate 3-isopropylmalate (64,65). Moreover, the ilvEBHC-leuABCD operon was remarkably up-regulated at 13 h at the transcriptional level (Fig.…”

Archaeal ApbC/Nbp35 Homologs Function as Iron-Sulfur Cluster Carrier Proteins

“…Grown aerobically at 37°C in Luria-Bertani Miller medium supplemented with ampicillin (100 g ml Ϫ1 ), these cultures were induced in mid-logarithmic-growth phase with 0.1% D-lactose. Cells were harvested and lysed as described previously, and the MMP0704 proteins were purified by nickel affinity chromatography using standard methods (13). Fractions containing the MMP0704-His 6 protein were combined in dialysis tubing (SpectraPor4; 14,000-molecular-weight cutoff) and dialyzed for 15 h in 2 liters of buffer containing 50 mM KCl and 20 mM Tris-HCl (pH 8.0) at 4°C.…”

“…Dialysis was continued for 5 h in fresh buffer before the protein inside the dialysis tubing was concentrated by dehydration with 20,000-Da polyethylene glycol. The MJ0283 proteins were purified by heat treatment of cell extracts prepared in a mixture of 50 mM KCl and 20 mM Tris-HCl (pH 8.0) (13). Total protein concentrations were determined by the Bradford dye-binding method (Thermo-Pierce) using bovine serum albumin as a standard.…”

“…Members of the Archaea produce many proteins that require iron-sulfur cluster cofactors, including redox proteins, aconitase-like dehydratases, radical S-adenosylmethionine enzymes, and RNA polymerase (9,13,18,32). Methanogenic archaea are obligate anaerobes, and many heterotrophic archaea grow anaerobically, indicating that oxidative stress has not limited the proliferation of iron-sulfur proteins in these lineages.…”

Cohesion Group Approach for Evolutionary Analysis of Aspartokinase, an Enzyme That Feeds a Branched Network of Many Biochemical Pathways

“…An alternative pathway from pyruvate (the citramalate pathway) requires fewer ATP equivalents and likely was the ancestral pathway. This pathway, first described in Leptospira (10) as a seemingly anomalous variation, is increasingly becoming recognized as either the favored route or the sole route to isoleucine biosynthesis in nature (23). Many organisms have the potential to utilize either pathway, with the citramalate pathway likely being the preferential route.…”