title: β-alanine biosynthesis in Methanocaldococcus jannaschii.
abstract: One efficient approach to assigning function to unannotated genes is to establish the enzymes that are missing in known biosynthetic pathways. One group of such pathways is those involved in coenzyme biosynthesis. In the case of the methanogenic archaeon Methanocaldococcus jannaschii as well as most methanogens, none of the expected enzymes for the biosynthesis of the β-alanine and pantoic acid moieties required for coenzyme A are annotated. To identify the gene(s) for β-alanine biosynthesis, we have established the pathway for the formation of β-alanine in this organism after experimentally eliminating other known and proposed pathways to β-alanine from malonate semialdehyde, l-alanine, spermine, dihydrouracil, and acryloyl-coenzyme A (CoA). Our data showed that the decarboxylation of aspartate was the only source of β-alanine in cell extracts of M. jannaschii. Unlike other prokaryotes where the enzyme producing β-alanine from l-aspartate is a pyruvoyl-containing l-aspartate decarboxylase (PanD), the enzyme in M. 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. A Km of ∼0.80 mM for l-aspartate with a specific activity of 0.09 μmol min(-1) mg(-1) at 70°C for the decarboxylation of l-aspartate was measured for the recombinant 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.
authors.affiliation: Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA.
authors.forename: Huimin
authors.initials: H
authors.lastname: Xu
authors.affiliation: Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA rhwhite@vt.edu.
authors.forename: Robert H
authors.initials: RH
authors.lastname: White
mesh_terms: D001224:Aspartic Acid / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D053898:Biosynthetic Pathways; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism; D003653:Decarboxylation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D007700:Kinetics; D063747:Methanocaldococcus / Q000235:genetics / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D017384:Sequence Deletion; D014445:Tyrosine Decarboxylase / Q000235:genetics / Q000378:metabolism; D015091:beta-Alanine / Q000032:analysis / Q000096:biosynthesis*
chemical_list: D001426:Bacterial Proteins; D015091:beta-Alanine; D001224:Aspartic Acid; D011732:Pyridoxal Phosphate; D002262:Carboxy-Lyases; C019747:aspartate 4-decarboxylase; D014445:Tyrosine Decarboxylase