BIOCHEMICAL AND GENETIC-CHARACTERIZATION OF A GENTISATE 1,2-DIOXYGENASE FROM SPHINGOMONAS SP. STRAIN RW5

A 4,103-bp long DNA fragment containing the structural gene of a genti sate 1,2-dioxygenase (EC 1.13.11.4), gtdA, from Sphingomonas sp. strai n 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 gtd A gene product with protein sequences in databases, including those of intradiol or extradiol ring-cleaving dioxygenases, revealed no signif icant homology except for a low similarity (27%) to the 1-hydroxy-2-na phthoate dioxygenase (phdI) of the phenanthrene degradation in Nocardi oides sp. strain KP7 (T. Iwabuchi and S. Harayama, J. Bacteriol. 179:6 488-6494, 1997). This gentisate 1,2-dioxygenase is thus a member of a new class of ring-cleaving dioxygenases. The gene was subcloned and hy perexpressed in E. coil. The resulting product was purified to homogen eity and partially characterized. Under denaturing conditions, the pol ypeptide 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 stoic hiometrically converted gentisate to maleylpyruvate, which was identif ied by gas chromatography-mass spectrometry analysis as its methyl est er. Values of affinity constants (K-m) and specificity constants (K-ca t/K-m) of the enzyme were determined to be 15 mu M and 511 s(-1) M-1 x 10(4) for gentisate and 754 mu M and 20 s(-1) M-1 x 10(4) for 3,6-dic hlorogentisate. Three further open reading frames (ORFs) were found do wnstream of gtdA. The deduced amino acid sequence of ORF 2 showed homo logy to several isomerases and carboxylases, and those of ORFs 3 and 4 exhibited significant homology to enzymes of the glutathione isomeras e superfamily and glutathione reductase superfamily, respectively.