This is the first report on the genetic analysis of PDC hydrolase, which is one of the protocatechuate 4,5-cleavage pathway enzymes. The PDC hydrolase gene of S. paucimobilis
SYK-6, designated ligI
, encodes a protein of 32,737 Da (293 amino acids). There was no similarity between the LigI amino acid sequence and those of the proteins in the databases, including the dienelactone hydrolase (4
) and the β-ketoadipate enol-lactone hydrolase (7
), which seemed to be functionally related to LigI.
gene is located approximately 5.4 kb upstream of ligA
. Interestingly, ligI
is transcribed divergently from ligAB
. This fact indicated that the PCA 4,5-cleavage pathway was composed of at least two distinct operons. We could not find other genes responsible for the PCA 4,5-cleavage pathway enzymes in the region sequenced. Downstream from ligI
, an incomplete ORF which had the same direction of transcription as ligI
was found. The predicted amino acid sequence of the product of this ORF showed significant similarity to those of the lignostilbene-α,β-dioxygenase (LSD) genes of Pseudomonas paucimobilis
). LSD has been reported to be a dioxygenase catalyzing the cleavage of the interphenyl double bond of lignostilbenes. The occurrence of the LSD gene homolog beside the PCA 4,5-cleavage pathway enzyme genes is interesting. Further nucleotide sequencing and functional analysis of the 10.5-kb Eco
RI fragment may address the functions of a putative LSD and other enzymes of the PCA 4,5-cleavage pathway.
The reaction product from PDC, catalyzed by LigI, was estimated to be two stereoisomers of CHM. OMA, which is a tautomer of CHM was not detected. The production of OMA from PDC was suggested by Maruyama (16
). OMA is an α-keto acid, and its TMS derivative is easily distinguishable in MS from that of CHM, because a keto group of OMA is not trimethylsilylated. OMA might have been degraded during the process of extraction, since α-keto acid is generally unstable.
The characteristics of the PDC hydrolases of S. paucimobilis
SYK-6, P. ochraceae
, and C. testosteroni
are summarized in Table . All enzymes are monomeric proteins. The molecular mass and pH optima of the SYK-6 enzyme are very similar to those of the P. ochraceae
enzyme. A higher affinity with CHM (or OMA) than with PDC and a higher Vmax
for PDC hydrolysis than for PDC synthesis were common features of the PDC hydrolases of SYK-6 and P. ochraceae
. Thiol reagents, such as Ellman’s reagent and N
-ethylmaleimide, strongly inhibited activity, suggesting that the cysteine residue is the catalytic site of these three enzymes. A similar inhibition was also observed in the P. ochraceae
and C. testosteroni
enzymes. In the α/β hydrolase fold enzymes, the catalytic nucleophile is located in a highly conserved peptide, Gly-X-(Ser/Cys)-X-Gly. The three-dimensional structure of the dienelactone hydrolase from Pseudomonas
sp. strain B13 was solved, and its catalytic nucleophile, cysteine residue 123, was shown to constitute part of a catalytic triad of residues (26
). Recently, Schrag and Cygler reported that a consensus sequence of these enzymes might better be described as Sm (small amino acid)-X-Nuc (nucleophile)-X-Sm, since the glycine residues at Nuc −2 and Nuc +2 are sometimes substituted for other small amino acids, including alanine and serine (32
). Among the three cysteine residues of LigI, Cys76 may be the catalytic site, since the sequence Ala74-Ser75-Cys76-His77-Gly78 corresponds to the consensus of Sm-X-Nuc-X-Sm.
Characteristics of PDC hydrolases of S. paucimobilis SYK-6, P. ochraceae, and C. testosteronia
gene insertion mutant, DLI, showed no PDC hydrolase activity when the DLI cells were grown on LB medium and on syringic acid. DLI was not able to grow on vanillic acid, and the cells of DLI grown on LB medium accumulated PDC from vanillic acid. These results indicated that the ligI
gene is unique in conferring PDC hydrolysis and is essential for growth of SYK-6 on vanillic acid. According to the proposed metabolic pathway of syringic acid in SYK-6, syringic acid is converted to PDC via 3-O
-methylgallic acid (11
). The production of 3-O
-methylgallic acid from syringic acid was evident from the results obtained with the ligH
gene. The mutation in the ligH
gene, the product of which is involved in the conversion of syringic acid to 3-O
-methylgallic acid, resulted in a growth defect on syringic acid (23
). Kersten et al. reported the production of PDC from 3-O
-methylgallic acid by 4,5-PCD (13
). However, the ligI
insertion mutant DLI grew on syringic acid and showed no PDC transformation activity and no PDC hydrolysis. These results obviously suggest that syringic acid is not metabolized via PDC and that neither 4,5-PCD nor LigI is involved in the metabolism of syringic acid.
The results obtained in this study strongly support the proposed PCA 4,5-cleavage pathway presented in Fig. . Further research is needed to elucidate the correct degradation pathway of syringic acid in SYK-6.