Purification and characterization of the coniferyl aldehyde dehydrogenase from Pseudomonas sp. Strain HR199 and molecular characterization of the gene

Abstract

The coniferyl aldehyde dehydrogenase (CALDH) of Pseudomonas sp. strain HR199 (DSM7063), which catalyzes the NAD+-dependent oxidation of coniferyl aldehyde to ferulic acid and which is induced during growth with eugenol as the carbon source, was purified and characterized. The native protein exhibited an apparent molecular mass of 86,000 +/- 5,000 Da, and the subunit mass was 49.5 +/- 2.5 kDa, indicating an alpha2 structure of the native enzyme. The optimal oxidation of coniferyl aldehyde to ferulic acid was obtained at a pH of 8.8 and a temperature of 26 degreesC. The Km values for coniferyl aldehyde and NAD+ were about 7 to 12 microM and 334 microM, respectively. The enzyme also accepted other aromatic aldehydes as substrates, whereas aliphatic aldehydes were not accepted. The NH2-terminal amino acid sequence of CALDH was determined in order to clone the encoding gene (calB). The corresponding nucleotide sequence was localized on a 9.4-kbp EcoRI fragment (E94), which was subcloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The partial sequencing of this fragment revealed an open reading frame of 1,446 bp encoding a protein with a relative molecular weight of 51,822. The deduced amino acid sequence, which is reported for the first time for a structural gene of a CALDH, exhibited up to 38.5% amino acid identity (60% similarity) to NAD+-dependent aldehyde dehydrogenases from different sources.

FIG. 1

CALDH, encoded by calB, catalyzes the NAD⁺-dependent oxidation of coniferyl aldehyde to ferulic acid.

FIG. 2

Results of SDS-PAGE of CALDH from Pseudomonas sp. strain HR199 grown in the presence of eugenol. Samples collected at various levels of purification were applied to an 11.5% (wt/vol) SDS-polyacrylamide gel as described in Materials and Methods. Lane 1, 46 μg of protein from the soluble fraction of the crude extract; lane 2, 34 μg of protein after chromatography on DEAE-Sephacel; lane 3, 4 μg of protein after chromatography on hydroxylapatite; lane 4, 2 μg of purified CALDH after chromatography on a Superdex 200 HiLoad column. The molecular masses of standard proteins (Stds) are given on the left in kilodaltons. Protein was stained with Serva Blue R.

FIG. 3

Homology of Pseudomonas sp. strain HR199 CALDH with ALDHs from other sources. The amino acid sequences of human liver mitochondrial ALDH (9) (row i), rat liver microsomal ALDH (21) (row iii), ALDH (alkH gene product) from Pseudomonas oleovorans (15) (row iv), vanillin dehydrogenase (vdh gene product) from Pseudomonas sp. strain HR199 (26) (row v), salicylaldehyde dehydrogenase (doxF gene product) from Pseudomonas sp. strain C18 (4) (row vi), benzaldehyde dehydrogenase (xylC gene product encoded by TOL plasmid pWW0) from P. putida (11) (row vii), succinic semialdehyde dehydrogenase (gabD gene product) from E. coli (22) (row viii), betaine ALDH (betB gene product) from E. coli (2) (row ix), and acetaldehyde dehydrogenase (acoD gene product) from A. eutrophus (25) (row x) were aligned with the CALDH amino acid sequence of Pseudomonas sp. strain HR199 deduced from calB (row ii). Amino acids are specified by standard one-letter abbreviations. Gaps (−) were introduced into the sequences in order to improve the alignment. Amino acid residues which are identical with those of the Pseudomonas sp. strain HR199 CALDH at particular sequence positions are shaded. Amino acids conserved across all 10 sequences are shown in bold. The consensus sequence is given in row xi.