-Alanine Biosynthesis in Methanocaldococcus jannaschii

One efficient approach to assigning function to unannotated genes is to establish the enzymes that are missing in known biosyn-thetic pathways. One group of such pathways is those involved in coenzyme biosynthesis. In the case of the methanogenic ar-chaeonMethanocaldococcus jannaschii as well as most methanogens, none of the expected enzymes for the biosynthesis of the -alanine and pantoic acid moieties required for coenzyme A are annotated. To identify the gene(s) for -alanine biosynthesis, we have established the pathway for the formation of -alanine in this organism after experimentally eliminating other known and proposed pathways to-alanine frommalonate semialdehyde, L-alanine, spermine, dihydrouracil, and acryloyl-coenzyme A (CoA). Our data showed that the decarboxylation of aspartate was the only source of -alanine in cell extracts ofM. jannaschii. Unlike other prokaryotes where the enzyme producing -alanine from L-aspartate is a pyruvoyl-containing L-aspartate decar-boxylase (PanD), the enzyme inM. jannaschii is a pyridoxal phosphate (PLP)-dependent L-aspartate decarboxylase encoded by MJ0050, the same enzyme that was found to decarboxylate tyrosine for methanofuran biosynthesis. AKm of0.80 mM for L-as-partate with a specific activity of 0.09mol min1 mg1 at 70°C for the decarboxylation of L-aspartate was measured for the re-combinant enzyme. The MJ0050 gene was also demonstrated to complement the Escherichia coli panD deletion mutant cells, in which panD encoding aspartate decarboxylase in E. coli had been knocked out, thus confirming the function of this gene in vivo. Coenzyme A (CoA) is an important coenzyme in all knownliving organisms where it functions as an acyl carrier for amide-, ester-, and thioester-forming reactions as well as activat