Genetic and biochemical characterization of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase and its role in the protocatechuate 4,5-cleavage pathway in Sphingomonas paucimobilis SYK-6

Abstract

Protocatechuate (PCA) is the key intermediate metabolite in the lignin degradation pathway of Sphingomonas paucimobilis SYK-6 and is metabolized to pyruvate and oxaloacetate via the PCA 4,5-cleavage pathway. We characterized the 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase gene (ligC). CHMS is the 4,5-cleavage product of PCA and is converted into 2-pyrone-4,6-dicarboxylate (PDC) by LigC. We found that ligC was located 295 bp downstream of ligB, which encodes the large subunit of the PCA 4,5-dioxygenase. The ligC gene consists of a 945-bp open reading frame encoding a polypeptide with a molecular mass of 34,590 Da. The deduced amino acid sequence of ligC showed 19 to 20% identity with 3-chlorobenzoate cis-dihydrodiol dehydrogenase of Alcaligenes sp. strain BR60 and phthalate cis-dihydrodiol dehydrogenases of Pseudomonas putida NMH102-2 and Burkholderia cepacia DBO1, which are unrelated to group I, II, and III microbial alcohol dehydrogenases (M. F. Reid and C. A. Fewson, Crit. Rev. Microbiol. 20:13-56, 1994). The ligC gene was expressed in Escherichia coli and LigC was purified to near homogeneity. Production of PDC from CHMS catalyzed by LigC was confirmed in the presence of NADP(+) by electrospray ionization-mass spectrometry and gas chromatography-mass spectrometry. LigC is a homodimer. The isoelectric point, optimum pH, and optimum temperature were estimated to be 5.3, 8.0, and 25 degrees C, respectively. The K(m) for NADP(+) was estimated to be 24.6 +/- 1.5 microM, which was approximately 10 times lower than that for NAD(+) (252 +/- 3.9 microM). The K(m)s for CHMS in the presence of NADP(+) and NAD(+) are 26.0 +/- 0.5 and 20.6 +/- 1.0 microM, respectively. Disruption of ligC in S. paucimobilis SYK-6 prevented growth with vanillate. Only PCA was accumulated during the incubation of vanillate with the whole cells of the ligC insertion mutant (DLC), indicating a lack of PCA 4,5-dioxygenase activity in DLC. However, the introduction of ligC into DLC restored its ability to grow on vanillate. PDC was suggested to be an inducer for ligAB gene expression.

FIG. 1

Catabolic pathway of vanillate for S. paucimobilis SYK-6 via the PCA 4,5-cleavage pathway. LigA and LigB, small and large subunits of PCA 4,5-dioxygenase (4,5-PCD) (27, 38); LigC, CHMS dehydrogenase (this study); LigI, PDC hydrolase (23); LigH, an essential gene product for vanillate and syringate O demethylations (26).

FIG. 2

Deletion analysis of the CHMS dehydrogenase gene and the insertional inactivation of ligC in S. paucimobilis SYK-6. (A) The CHMS dehydrogenase activities of the cells harboring each subclone are presented. The small arrows indicate the direction of transcription from the lac (P_lac) or T7 (P_T7) promoters. Large filled arrows indicate the PCA 4,5-cleavage pathway genes, ligI, ligA, ligB, and ligC. A partly filled large arrow represents part of the ORF of the lignostilbene α,β-dioxygenase homolog (lsdA). E. coli JM109, E. coli BL21(DE3), and P. putida PpY1100 were used as host strains for pHN139F and pHN139R; pHNC2 and pSM21; and pVA01, pVAD2, and pVAD4, respectively. E, EcoRI; P, PstI; Sc, SacI; Sl, SalI; Sm, SmaI; Xb, XbaI; Xh, XhoI. (B) Schematic representation of the insertional inactivation of ligC by the kanamycin resistance gene from pUC4K. Bs, BsmI; E, EcoRI; P, PstI; Sc, SacI; Sl, SalI; Sm, SmaI; Xb, XbaI; Xh, XhoI.

FIG. 3

Identification of the reaction product from CHMS catalyzed by LigC. (A) Gas chromatogram of the TMS derivative of the reaction product from CHMS catalyzed by purified LigC. CHMS (100 μM) was incubated with 3 μg of purified LigC for 2 min in the presence of 200 μM NADP⁺. The reaction product was extracted by ethylacetate and trimethylsilylated (TMS). (B) Mass spectrum of the TMS derivative of the product with a retention time of 27.8 min shown in panel A. (C) Mass spectrum of the authentic TMS-PDC.

FIG. 4

Identification of the metabolite accumulated in DLC culture from vanillate. Vanillate was incubated with the whole cells of DLC for 12 h. The negative-ion ESI-MS spectrum of a portion of the supernatant of a reaction mixture is shown. The detected ion (II) at m/z 153.0 corresponded to the deprotonated molecular ion of PCA and was a major product from vanillate (ion I). The ion at m/z 185.0 representing CHMS was not detected.