Biochemical and genetic characterization of a gentisate 1, 2-dioxygenase from Sphingomonas sp. strain RW5

Abstract

A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC 1.13.11.4), gtdA, from Sphingomonas sp. strain RW5 was cloned and sequenced. The gtdA gene encodes a 350-amino-acid polypeptide with a predicted size of 38.85 kDa. Comparison of the gtdA gene product with protein sequences in databases, including those of intradiol or extradiol ring-cleaving dioxygenases, revealed no significant homology except for a low similarity (27%) to the 1-hydroxy-2-naphthoate dioxygenase (phdI) of the phenanthrene degradation in Nocardioides sp. strain KP7 (T. Iwabuchi and S. Harayama, J. Bacteriol. 179:6488-6494, 1997). This gentisate 1,2-dioxygenase is thus a member of a new class of ring-cleaving dioxygenases. The gene was subcloned and hyperexpressed in E. coli. The resulting product was purified to homogeneity and partially characterized. Under denaturing conditions, the polypeptide exhibited an approximate size of 38.5 kDa and migrated on gel filtration as a species with a molecular mass of 177 kDa. The enzyme thus appears to be a homotetrameric protein. The purified enzyme stoichiometrically converted gentisate to maleylpyruvate, which was identified by gas chromatography-mass spectrometry analysis as its methyl ester. Values of affinity constants (Km) and specificity constants (Kcat/Km) of the enzyme were determined to be 15 microM and 511 s-1 M-1 x 10(4) for gentisate and 754 microM and 20 s-1 M-1 x 10(4) for 3, 6-dichlorogentisate. Three further open reading frames (ORFs) were found downstream of gtdA. The deduced amino acid sequence of ORF 2 showed homology to several isomerases and carboxylases, and those of ORFs 3 and 4 exhibited significant homology to enzymes of the glutathione isomerase superfamily and glutathione reductase superfamily, respectively.

FIG. 1

Nucleotide and deduced amino acid sequence of the putative 1,053-bp gene gtdA. The sense DNA strand is shown. The putative Shine-Dalgarno sequence is underlined. A point indicates the stop codon.

FIG. 2

SDS-PAGE analysis of purified GDO from hyperexpressed gtdA. Protein samples were separated by electrophoresis on a 12% polyacrylamide gel and stained with Coomassie blue. Lane 1, protein standards (in kilodaltons); lane 2, E. coli BL21 (DE3, pJW48) cell extract from induced whole cells; lane 3, GDO fraction from fast-flow DEAE-Sepharose chromatography; lane 4, GDO fraction from Mono Q separation; lane 5, GDO fraction after gel filtration.

FIG. 3

Mass spectrum (70eV) of trimethylsulfonium hydroxide-derivatized maleylpyruvate.