Functional characterization of an aminotransferase required for pyoverdine siderophore biosynthesis in Pseudomonas aeruginosa PAO1.
The fluorescent dihydroxyquinoline chromophore of the pyoverdine siderophore in Pseudomonas is a condensation product of D-tyrosine and l-2,4-diaminobutyrate. Both pvdH and asd (encoding aspartate beta-semialdehyde dehydrogenase) knockout mutants of Pseudomonas aeruginosa PAO1 were unable to synthesize pyoverdine under iron-limiting conditions in the absence of l-2,4-diaminobutyrate in the culture media. The pvdH gene was subcloned, and the gene product was hyperexpressed and purified from P. aeruginosa PAO1. PvdH was found to catalyze an aminotransferase reaction, interconverting aspartate beta-semialdehyde and l-2,4-diaminobutyrate. Steady-state kinetic analysis with a novel coupled assay established that the enzyme adopts a ping-pong kinetic mechanism and has the highest specificity for alpha-ketoglutarate. The specificity of the enzyme toward the smaller keto acid pyruvate is 41-fold lower. The enzyme has negligible activity toward other keto acids tested. Homologues of PvdH were present in the genomes of other Pseudomonas spp. These homologues were found in the DNA loci of the corresponding genomes that contain other pyoverdine synthesis genes. This suggests that there is a general mechanism of l-2,4-diaminobutyrate synthesis in Pseudomonas strains that produce the pyoverdine siderophore.
Department of Microbiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Chris S
CS
Vandenende
Matthew
M
Vlasschaert
Stephen Y K
SY
Seah
D000613:Aminobutyrates / Q000378:metabolism; D001223:Aspartate-Semialdehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D003001:Cloning, Molecular; D017353:Gene Deletion; D005798:Genes, Bacterial; D007656:Ketoglutaric Acids / Q000378:metabolism; D016254:Mutagenesis, Insertional; D009154:Mutation; D009842:Oligopeptides; D010860:Pigments, Biological / Q000096:biosynthesis*; D011549:Pseudomonas; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D017385:Sequence Homology; D013379:Substrate Specificity; D026801:Synteny; D000637:Transaminases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D000613:Aminobutyrates; D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; D009842:Oligopeptides; D010860:Pigments, Biological; C005959:2,4-diaminobutyric acid; D001224:Aspartic Acid; D014443:Tyrosine; C006644:aspartic semialdehyde; C042453:pyoverdin; D001223:Aspartate-Semialdehyde Dehydrogenase; D000637:Transaminases