[Show abstract][Hide abstract] ABSTRACT: Catechol 2,3-dioxygenase (C23O; EC 126.96.36.199), exemplified by XylE and NahH, catalyzes the ring cleavage of catechol and some substituted catechols. C23O is inactivated at an appreciable rate during the ring cleavage of 4-methylcatechol due to the oxidation of the Fe(II) cofactor to Fe(III). In this study, a C23O exhibiting improved activity against 4-methylcatechol was isolated. To isolate this C23O, diverse C23O gene sequences were PCR amplified from DNA which had been isolated from mixed cultures of phenol-degrading bacteria and subcloned in the middle of a known C23O gene sequence (xylE or nahH) to construct a library of chimeric C23O genes. These chimeric C23O genes were then introduced into Pseudomonas putida possessing some of the toluene catabolic genes (xylXYZLGFJQKJI). When a C23O gene (e.g., xylE) is introduced into this strain, the transformants cannot generally grow on p-toluate because 4-methylcatechol, a metabolite of p-toluate, is a substrate as well as a suicide inhibitor of C23O. However, a transformant of this strain capable of growing on p-toluate was isolated, and a chimeric C23O (named NY8) in this transformant was characterized. The rate of enzyme inactivation by 4-methylcatechol was lower in NY8 than in XylE. Furthermore, the rate of the reactivation of inactive C23O in a solution containing Fe(II) and ascorbic acid was higher in NY8 than in XylE. These properties of NY8 might allow the efficient metabolism of 4-methylcatechol and thus allow host cells to grow on p-toluate.
Applied and Environmental Microbiology 04/2004; 70(3):1804-10. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 3-Chlorobiphenyl is known to be mineralized by biphenyl-utilizing bacteria to 3-chlorobenzoate, which is further metabolized to 3-chlorocatechol. An extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHB12O; EC 188.8.131.52), which is encoded by the bphC gene, catalyzes the third step of the upper pathway of 3-chlorobiphenyl degradation. In this study, two full-length bphCs and nine partial fragments of bphCs fused to the 3' end of bphC in Pseudomonas pseudoalcaligenes KF707 were cloned from different biphenyl-utilizing soil bacteria and expressed in Escherichia coli. The enzyme activities of the expressed DHB12Os were inhibited to varying degrees by 3-chlorocatechol, and the E. coli cells overexpressing DHB12O could not grow or grew very slowly in the presence of 3-chlorocatechol. These sensitivities of enzyme activity and cell growth to 3-chlorocatechol were well correlated, and this phenomenon was employed in screening chimeric BphCs formed by family shuffling of bphC genes isolated from Comamonas testosteroni KF704 and C. testosteroni KF712. The resultant DHB12Os were more resistant by a factor of two to 3-chlorocatechol than one of the best parents, KF707 DHB12O.
Journal of Biochemistry 04/2004; 135(3):305-17. · 3.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Catechol 2,3-dioxygenases (C23Os; EC 184.108.40.206) form a large protein family that is divided into several subgroups. Amino acid sequences of C23Os belonging to subgroup I.2.A and those belonging to I.2.B are found to be approx. 50% identical. When the central parts of the C23O sequences belonging to I.2.B were fused with the N-terminal and C-terminal sequences of I.2.A C23O, the hybrid enzymes were not active. To understand why these hybrid C23Os were inactive, hybrids between XylE(P) (C23O found in a Pseudomonas strain; subgroup I.2.A) and XylE(S) (C23O found in a Sphingomonas strain; subgroup I.2.B) were constructed. HB3-C23O consisted mostly of the XylE(S) sequence, except that its C-terminal end was derived from XylE(P). While HB3-C23O was not active, HB4-C23O, carrying shorter C-terminal XylE(P) sequences than HB3-C23O, was active. This observation indicated that certain amino acid residues at the C-terminus were crucial for C23O activity in the hybrid forms of enzymes between XylE(P) and XylE(S). According to the crystal structure of XylE(P), the C-terminal region is involved in the formation of a quaternary structure. Amino acid differences between HB3-C23O and HB4-C23O included the specific beta-strand for oligomerization. Thus the quaternary structures of active C23Os, XylE(S), XylE(P) and HB4-C23O, as well as that of inactive HB3-C23O, were examined. Active enzymes XylE(S), XylE(P) and HB4-C23O were homotetrameric, while HB3-C23O existed only as a monomer. We concluded that hybrids of subgroups I.2.A and I.2.B were often inactive because of a defect in their oligomerization.
[Show abstract][Hide abstract] ABSTRACT: A method was developed to isolate central segments of catechol 2,3-dioxygenase (C23O) genes from environmental samples and to insert these C23O gene segments into nahH (the structural gene for C23O encoded by catabolic plasmid NAH7) by replacing the corresponding nahH sequence with the isolated segments. To PCR-amplify the central C23O gene segments, a pair of degenerate primers was designed from amino acid sequences conserved among C23Os. Using these primers, central regions of the C23O genes were amplified from DNA isolated from a mixed culture of phenol-degrading or crude oil-degrading bacteria. Both the 5′ and 3′ regions of nahH were also PCR-amplified by using appropriate primers. These three PCR products, the 5′-nahH and 3′-nahH segments and the central C23O gene segments, were mixed and PCR-amplified again. Since the primers for the amplification of the central C23O gene segments were designed so that the 20 nucleotides at both ends of the segments are identical to the 3′ end of the 5′-nahH segment and the 5′ end of the 3′-nahH segment, respectively, the central C23O gene segments could anneal to both the 5′- and 3′-nahH segments. After the second PCR, hybrid C23O genes in the form of (5′-nahH segment—central C23O gene segment—3′-nahH segment) were amplified to full length. The resulting products were cloned into a vector and used to transform Escherichia coli. This method enabled divergent C23O sequences to be readily isolated, and more than 90% of the hybrid plasmids expressed C23O activity. Thus, the present method is useful to create, without isolating bacteria, a library of functional hybrid genes.