A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC 1.13.11.4 ), gtdA, fromSphingomonas sp. strain RW5 was cloned and sequenced. ThegtdA 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 (K cat/K m) of the enzyme were determined to be 15 μM and 511 s−1M−1 × 104 for gentisate and 754 μM and 20 s−1 M−1 × 104 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.

SynopsisThe structure inducing properties of L-leucine, L-isoleucine, and L-norleucine residues incorporated into ply@-lysine) were investigated by the observation of the circular dichroism of the respective random copolypeptides. The comparison involves the coil-helix transition in water/methanol mixtures, the formation of ordered structures at higher pH, and the kinetics of the a-helix to &conformation transition of the leucine and norleucine copolymers induced by temperature changes a t pH 10.5. The results confirm the known properties of the leucine residue, strongly supporting the a-helix conformation. They also support the idea that the isoleucine residue is one of the most powerful candidates for S-structure formation, and they show that the unbranched norleucine residue has intermediate properties. The results are discussed on the basis of steric and hydrophobic properties of the three side chains.

No abstract

No abstract

No abstract