A 2-oxoacid dehydrogenase complex of Haloferax volcanii is essential for growth on isoleucine but not on other branched-chain amino acids

Abstract

The halophilic archaeon Haloferax volcanii contains three operons encoding 2-oxoacid dehydrogenase complexes (OADHCs) OADHC1-OADHC3. However, the biological role of these OADHCs is not known as previous studies have demonstrated that they cannot use any of the known OADHC substrates. Even the construction of single mutants in all three oadhc operons, reported recently, could not identify a substrate. Therefore, all three possible double mutants and a triple mutant were generated, and single, double and triple mutants were compared to the wild-type. The four mutants devoid of a functional OADHC1 had a reduced growth yield during nitrate-respirative growth on tryptone. A metabolome analysis of the medium after growth of the triple mutant in comparison to the wild-type revealed that the mutant was unable to degrade isoleucine and leucine, in contrast to the wild-type. It was shown that oadhc1 mutants were unable to grow in synthetic medium on isoleucine, in contrast to the other mutants and the isogenic parent strain. However, all strains grew indistinguishably on valine and leucine. The transcript of the oadhc1 operon was highly induced during growth on isoleucine. However, attempts to detect enzymic activity were unsuccessful, while the branched-chain OADHC (BCDHC) of Pseudomonas putida could be measured easily. Therefore, the growth capability of the triple mutant and the wild-type on the two first degradation intermediates of isoleucine was tested and provided further evidence that OADHC is involved in isoleucine degradation. Taken together, the results indicate that OADHC1 is a specialized BCDHC that uses only one (or maximally two) of the three branched-chain 2-oxoacids, in contrast to BCDHCs from other species.

Microbial ecology of an Antarctic hypersaline lake: genomic assessment of ecophysiology among dominant haloarchaea

“…In addition, the DL1 genome was statistically overrepresented for COGs associated with BCAA uptake and catabolism, including OADHC components (Table 1, Supplementary Table S3). In archaea, certain OADHCs can catalyze the decarboxylation of branched-chain 2-oxoacids that result from BCAA catabolism, as well as pyruvate (Heath et al, 2004(Heath et al, , 2007Sisignano et al, 2010). Thus, these COGs associated with general amino acid metabolism, BCAA uptake and catabolism, and OADHC components, likely describe a common substrate preference by DL1.…”

Functional Genomic and Advanced Genetic Studies Reveal Novel Insights into the Metabolism, Regulation, and Biology ofHaloferax volcanii

“…volcanii harbors three gene clusters that encode subunits of three 2-oxoacid dehydrogenase complexes, but the substrates and biological roles for all three enzyme complexes remained obscure for a long time [67–72]. In an attempt to apply genetics to solve the question, seven strains were constructed that contained the three possible single gene deletions, the three possible double deletions, and a triple gene deletion [73]. Comparison of the extracellular metabolomes of wild-type and mutants revealed that the wild type depleted complex media of isoleucine, while mutants lacking one of the 2-oxoacid dehydrogenase complexes (OADHC-1) were unable to do so.…”

“…Comparison of the extracellular metabolomes of wild-type and mutants revealed that the wild type depleted complex media of isoleucine, while mutants lacking one of the 2-oxoacid dehydrogenase complexes (OADHC-1) were unable to do so. Subsequent analyses verified that OADHC-1 is indeed essential for degrading isoleucine but not the other branched-chain amino acids [73]. …”

Conserved active site cysteine residue of archaeal THI4 homolog is essential for thiamine biosynthesis in Haloferax volcanii

“…Hfx. volcan ii is also demonstrated to synthesize key metabolic enzymes that are TPP-dependent including the 2-oxoacid (α-ketoacid): ferredoxin oxidoreductases used to mediate the oxidative decarboxylation of pyruvate and 2-oxoglutarate [24] and three 2-oxoacid dehydrogenases used under nitrate-respirative conditions [25] including one involved in growth on isoleucine [26]. The ΔubaA mutant required to analyze the ThiS/ThiF-type pathway was already available from previous study [11].…”