Cloning and sequencing of the Sphingomonas (Pseudomonas) paucimobilis gene essential for the O demethylation of vanillate and syringate

Abstract

Sphingomonas (Pseudomonas) paucimobilis SYK-6 is able to grow on 5,5'-dehydrodivanillic acid (DDVA), syringate, vanillate, and other dimeric model compounds of lignin as a sole carbon source. Nitrosoguanidine mutagenesis of S. paucimobilis SYK-6 was performed, and two mutants with altered DDVA degradation pathways were isolated. The mutant strain NT-1 could not degrade DDVA, but could degrade syringate, vanillate, and 2,2',3'-trihydroxy-3-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA). Strain DC-49 could slowly assimilate DDVA, but could degrade neither vanillate nor syringate, although it could degrade protocatechuate and 3-O-methylgallate. A complementing DNA fragment of strain DC-49 was isolated from the cosmid library of strain SYK-6. The minimum DNA fragment complementing DC-49 was determined to be the 1.8-kbp insert of pKEX2.0. Sequencing analysis showed an open reading frame of 1,671 bp in this fragment, and a similarity search indicated that the deduced amino acid sequence of this open reading frame had significant similarity (60%) to the formyltetrahydrofolate synthetase of Clostridium thermoaceticum.

FIG. 1

Degradation pathway of various lignin-related compounds in S. (Pseudomonas) paucimobilis SYK-6 (14). The O demethylation steps of phenylmethylether are represented by solid arrows. The enzymes catalyzing each step are indicated on the figure as follows: Lig A, B, protocatechuate 4,5-dioxygenase small and large subunits, respectively (27); Lig C, 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (26); Lig D, arylglycerol-β-aryl ether Cα dehydrogenase (20); Lig F(E), α-keto-α-arylglycerol-β-aryl ether β-etherase isozymes (21, 22); Lig Z, OH-DDVA dioxygenase (11). OH-DDVA is converted to 5-carboxyvanillate with LigZ and LigY. Lig H, an enzyme related to the O demethylation of vanillate and syringate (this study). 5-Carboxyvanillate decarboxylase (represented by dotted arrow a) activity was detected in S. paucimobilis SYK-6 cell extract (24), and 3-O-methylgallate was detected by the metabolic experiment with 5-carboxyvanillate (represented by dotted arrow b) (14). The 2-pyrone-4,6-dicarboxylic acid metabolic pathway is represented according to findings from previous studies (18, 19, 26, 31). CHA aldolase, 4-carboxy-4-hydroxy-2-adipic acid aldolase; TCA cycle, tricarboxylic acid cycle.

FIG. 2

Deletion analysis of complementation ability for vanillate and syringate degradation of strain DC-49. The plasmid construction is indicated in Table 1. The degradation ability is represented according to Table 2. The location and direction of the ligH gene are indicated by an arrow.

FIG. 3

Amino acid alignment between LigH and formyltetrahydrofolate synthetase (FTHS) of C. thermoaceticum (16). Identical and similar amino acids are indicated by asterisks and colons, respectively. Residues proposed as putative ATP binding regions in FTHS of C. thermoaceticum are in boldface (16). Some important conserved regions in FTHSs suggested by PROSITE database (3) under accession no. PS00721 and PS00722 are boxed.

FIG. 4

Detection of the ligH gene product. Total cellular proteins of strains grown in LB medium were electrophoresed. Lane 1, MV1190(pUC119); lane 2, MV1190(pUEX2.0u) plus IPTG.