Functional characterization of an aminotransferase required for pyoverdine siderophore biosynthesis in Pseudomonas aeruginosa PAO1

Abstract

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.

FIG. 1.

Structure of pyoverdine produced by P. aeruginosa PAO1 (41). Carbon atoms of the chromophore derived from l-2,4-diaminobutyrate are numbered. R indicates a variable acyl group.

FIG. 2.

Locus of P. aeruginosa PAO1 genome containing the pvdH gene (PA2413). The promoters of pvdH and PA2412, indicated by vertical lines, contain iron starvation box consensus sequences, suggesting that they are both regulated by the sigma factor, PvdS (21).

FIG. 3.

Spectra of culture supernatants of pvdH (A) and asd (B) knockout mutants grown in CAA media. The dashed lines are the spectra for culture supernatants of mutants grown in medium containing l-2,4-diaminobutyrate, and the solid lines are spectra for culture supernatants of mutants grown in medium without l-2,4-diaminobutyrate.

FIG. 4.

Metabolic pathway showing the relationship between the reaction catalyzed by PvdH and aspartate β-semialdehyde dehydrogenase (Asd). The enzymatic steps required to convert aspartate β-semialdehyde to meso-diaminopimelate in P. aeruginosa have not been elucidated.

FIG. 5.

Coomassie blue-stained SDS-PAGE gel of purified PvdH. The gel was loaded with samples of PvdH from the crude extract (lane 2), the preparation after anion exchange (lane 3), and the preparation after gel filtration (lane 4). The molecular masses of the proteins in the standard (lane 1) are indicated on the left.

FIG. 6.

Lineweaver-Burk and secondary plots of PvdH activities with l-2,4-diaminobutyrate and α-ketoglutarate as substrates. (A) Lineweaver-Burk plot showing the relationship of PvdH activity and l-2,4-diaminobutyrate concentration. The α-ketoglutarate concentration was fixed at 100 μM (•), 150 μM (○), 200 μM (▾), 300 μM (▿), or 1 mM (▪). (B) y intercepts from the Lineweaver-Burk graph plotted against the reciprocal of the α-ketoglutarate concentration.