Characterization of the 4-carboxy-4-hydroxy-2-oxoadipate aldolase gene and operon structure of the protocatechuate 4,5-cleavage pathway genes in Sphingomonas paucimobilis SYK-6

Abstract

The protocatechuate (PCA) 4,5-cleavage pathway is the essential metabolic route for degradation of low-molecular-weight products derived from lignin by Sphingomonas paucimobilis SYK-6. In the 10.5-kb EcoRI fragment carrying the genes for PCA 4,5-dioxygenase (ligAB), 2-pyrone-4,6-dicarboxylate hydrolase (ligI), 4-oxalomesaconate hydratase (ligJ), and a part of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (ligC), we found the ligK gene, which encodes 4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase. The ligK gene was located 1,183 bp upstream of ligI and transcribed in the same direction as ligI. We also found the ligR gene encoding a LysR-type transcriptional activator, which was located 174 bp upstream of ligK. The ligK gene consists of a 684-bp open reading frame encoding a polypeptide with a molecular mass of 24,131 Da. The deduced amino acid sequence of ligK showed 57 to 88% identity with those of the corresponding genes recently reported in Sphingomonas sp. strain LB126, Comamonas testosteroni BR6020, Arthrobacter keyseri 12B, and Pseudomonas ochraceae NGJ1. The ligK gene was expressed in Escherichia coli, and the gene product (LigK) was purified to near homogeneity. Electrospray-ionization mass spectrometry indicated that LigK catalyzes not only the conversion of CHA to pyruvate and oxaloacetate but also that of oxaloacetate to pyruvate and CO(2). LigK is a hexamer, and its isoelectric point is 5.1. The K(m) for CHA and oxaloacetate are 11.2 and 136 micro M, respectively. Inactivation of ligK in S. paucimobilis SYK-6 resulted in the growth deficiency of vanillate and syringate, indicating that ligK encodes the essential CHA aldolase for catabolism of these compounds. Reverse transcription-PCR analysis revealed that the PCA 4,5-cleavage pathway genes of S. paucimobilis SYK-6 consisted of four transcriptional units, including the ligK-orf1-ligI-lsdA cluster, the ligJAB cluster, and the monocistronic ligR and ligC genes.

FIG. 1.

Catabolic pathway of vanillate and syringate by S. paucimobilis SYK-6 (A) and organization of the PCA 4,5-cleavage pathway genes (B). (A) LigA and LigB, the small and large subunits of 4,5-PCD (31, 44); LigH, an essential gene product for vanillate and syringate O demethylations (30); LigC, CHMS dehydrogenase (27); LigI, PDC hydrolase (28); LigJ, OMA hydratase (11); LigK, CHA aldolase/oxaloacetate (OA) decarboxylase (in this study). The degradation pathway for syringate indicated by a dashed line was suggested in our previous study (11, 27, 28). (B) orf1, orf2, ligI, ligJ, ligK, ligA, ligB, ligC, ligR, and lignostilbene α,β-dioxygenase homolog (lsdA) genes are demonstrated by the filled arrows. Vertical bars above the restriction map indicate the positions of the Km^r gene insertion of orf1 mutant (DF1), ligK mutant (DLK), ligR mutant (DLR), and orf2 mutant (DF2). Double-headed arrows indicate locations of amplified RT-PCR products shown in Fig. 5. Abbreviations for restriction enzymes: C, ClaI; E, EcoRI; Ec, Eco47III; P, PstI; Pu, PpuMI; Sh, SphI; Sl, SalI; Sm, SmaI; X, XhoI; Xb, XbaI.

FIG. 2.

Identification of the reaction product from CHA catalyzed by LigK. CHA (200 μM) was incubated with 0.5 μg of LigK in 0.1 M Tris-acetate buffer (pH 8.0) containing 1 mM MgCl₂. (A) Negative-ion ESI-MS spectrum of the reaction mixture without CHA. (B to D) Negative-ion ESI-MS spectrum of the reaction mixture at 0, 1, and 5 min of incubation with LigK, respectively.

FIG. 3.

Identification of accumulated products from vanillate and syringate by DLK. (A and B) Gas chromatograms of trimethylsilylated derivatives of the accumulated products from vanillate and syringate, respectively. In both cultures, PDC, the enol form of OMA, product I, and unidentified product II were observed. (C and D) Negative-ion ESI-MS spectrum of the same reaction mixture used in panels A and B, respectively.

FIG. 4.

Growth of SYK-6 and ligR insertion mutant (DLR) on vanillate and syringate. Growth of SYK-6 (open circles) and DLR (filled circles) on 10 mM vanillate (A) and 10 mM syringate (B). The results are the means of the representative of three independent experiments.

FIG. 5.

Agarose gel electrophoresis of RT-PCR products amplified from SYK-6 cells grown on vanillate. The sizes of molecular weight markers in lane M are indicated on the left side of the gels. Odd-numbered lanes are controls without reverse transcriptase. The primers for spanning each ORF intergenic region or ORF internal region are shown in the Materials and Methods. Lanes: 1 and 2, lsdA-ligI intergenic region (expected size, 581 bp); 3 and 4, ligI-orf1 intergenic region (expected size, 491 bp); 5 and 6, orf1-ligK intergenic region (expected size, 271 bp); 7 and 8, ligR-orf2 intergenic region (expected size, 726 bp); 9 and 10, orf2-ligJ intergenic region (expected size, 427 bp); 11 and 12, ligJ-ligA intergenic region (expected size, 520 bp); 13 and 14, ligA-ligB intergenic region (expected size, 564 bp); 15 and 16, ligB-ligC intergenic region (expected size, 489 bp); 17 and 18, ligC internal region (expected size, 509 bp); 19 and 20, ligR internal region (expected size, 622 bp); and 21 and 22, orf2 internal region (expected size, 653 bp).