A Chemoenzymatic Formal Synthesis of Epoxyquinols A and B

A formal synthesis of epoxyquinols A and B was completed in nine steps starting from benzoic acid. Enantioselectivity was established through an enzymatic arene dihydroxylation reaction performed by whole cells of Ralstonia eutropha B9 expressing benzoate dioxygenase. Subsequent formation of the enone core was facilitated by a Dauben–Michno-type oxidative transposition of an allylic tertiary alcohol.

Diversity of the gene of benzoate dioxygenase in bacterial associations isolated from long term organochlorine-contaminated soils

Background. Communities of bacteria with specific enzymes are formed in the soil with long-term organochlorine contamination. The aim of this study was to analyze the diversity of the benA gene encoding the -subunit of the benzoate 1,2-dioxygenase in aerobic bacterial associations isolated from the soils of the Chapayevsk-city (Samara region, Russia). Materials and methods. The soil samples were taken on the territory, contaminated with organochlorine compounds for a long time. As a selection factor in the enrichment cultures were used 4-chlorobenzoic acid and chlorobenzene, in the pure cultures benzoic acid. The isolation of total DNA from bacterial associations was performed using a commercial FastDNA Spin Kit for Soil kit (USA). Amplification was performed on a MyCycler instrument (USA). Determination of the nucleotide sequence was performed on an automatic sequencer Genetic Analyzer 3500XL (USA). The search and analysis for benA gene homologs was carried out using international GenBank databases and BLAST system (http://www.ncbi.nlm.nih.gov). Results. As a result of selection, 12 associations of aerobic bacteria were obtained. Fragments of the benA gene (-subunit of benzoate dioxygenase) were obtained with the total DNA of six bacterial associations selected on chlorobenzene and with the total DNA of three bacterial associations selected on 4-chlorobenzoate. Pure cultures of aerobic bacterial strains using benzoic acid as a carbon source were isolated from benA-positive associations. It was established that the amplified fragments with the DNA of the A1, A4, A5, B1, B2, B3, B4 and B6 association strains form a single phylogenetic cluster with the -subunit gene of the benzoate dioxygenase of the Pseudomonas putida strain KT2440 (level of similarity is 9698%). The amplified fragment with the DNA of strain B5-170 (association B5) forms a cluster with the gene of the -subunit of the benzoate dioxygenase of the strain Pseudomonas sp. VLB120 (93% similarity).

Benzoate dioxygenase fromRalstonia eutrophaB9 – unusual regiochemistry of dihydroxylation permits rapid access to novel chirons

Oxidation of benzoic acid by a microorganism expressing benzoate dioxygenase leads to the formation of an unusualipso,orthoarenecis-diol in sufficient quantities to be useful for synthesis.

Characterization of Hybrid Toluate and Benzoate Dioxygenases

ABSTRACT Toluate dioxygenase of Pseudomonas putida mt-2 (TADOmt2) and benzoate dioxygenase of Acinetobacter calcoaceticus ADP1 (BADOADP1) catalyze the 1,2-dihydroxylation of different ranges of benzoates. The catalytic component of these enzymes is an oxygenase consisting of two subunits. To investigate the structural determinants of substrate specificity in these ring-hydroxylating dioxygenases, hybrid oxygenases consisting of the α subunit of one enzyme and the β subunit of the other were prepared, and their respective specificities were compared to those of the parent enzymes. Reconstituted BADOADP1 utilized four of the seven tested benzoates in the following order of apparent specificity: benzoate > 3-methylbenzoate > 3-chlorobenzoate > 2-methylbenzoate. This is a significantly narrower apparent specificity than for TADOmt2 (3-methylbenzoate > benzoate ∼ 3-chlorobenzoate > 4-methylbenzoate ∼ 4-chlorobenzoate ≫ 2-methylbenzoate ∼ 2-chlorobenzoate [Y. Ge, F. H. Vaillancourt, N. Y. Agar, and L. D. Eltis, J. Bacteriol. 184:4096-4103, 2002]). The apparent substrate specificity of the αBβT hybrid oxygenase for these benzoates corresponded to that of BADOADP1, the parent from which the α subunit originated. In contrast, the apparent substrate specificity of the αTβB hybrid oxygenase differed slightly from that of TADOmt2 (3-chlorobenzoate > 3-methylbenzoate > benzoate ∼ 4-methylbenzoate > 4-chlorobenzoate > 2-methylbenzoate > 2-chlorobenzoate). Moreover, the αTβB hybrid catalyzed the 1,6-dihydroxylation of 2-methylbenzoate, not the 1,2-dihydroxylation catalyzed by the TADOmt2 parent. Finally, the turnover of this ortho-substituted benzoate was much better coupled to O2 utilization in the hybrid than in the parent. Overall, these results support the notion that the α subunit harbors the principal determinants of specificity in ring-hydroxylating dioxygenases. However, they also demonstrate that the β subunit contributes significantly to the enzyme's function.

Cloning and Expression of the Benzoate Dioxygenase Genes from Rhodococcus sp. Strain 19070

ABSTRACT The bopXYZ genes from the gram-positive bacteriumRhodococcus sp. strain 19070 encode a broad-substrate-specific benzoate dioxygenase. Expression of the BopXY terminal oxygenase enabled Escherichia coli to convert benzoate or anthranilate (2-aminobenzoate) to a nonaromaticcis-diol or catechol, respectively. This expression system also rapidly transformed m-toluate (3-methylbenzoate) to an unidentified product. In contrast, 2-chlorobenzoate was not a good substrate. The BopXYZ dioxygenase was homologous to the chromosomally encoded benzoate dioxygenase (BenABC) and the plasmid-encoded toluate dioxygenase (XylXYZ) of gram-negative acinetobacters and pseudomonads. Pulsed-field gel electrophoresis failed to identify any plasmid inRhodococcus sp. strain 19070. Catechol 1,2- and 2,3-dioxygenase activity indicated that strain 19070 possesses bothmeta- and ortho-cleavage degradative pathways, which are associated in pseudomonads with the xyl andben genes, respectively. Open reading frames downstream ofbopXYZ, designated bopL and bopK, resembled genes encoding cis-diol dehydrogenases and benzoate transporters, respectively. The bop genes were in the same order as the chromosomal ben genes of P. putida PRS2000. The deduced sequences of BopXY were 50 to 60% identical to the corresponding proteins of benzoate and toluate dioxygenases. The reductase components of these latter dioxygenases, BenC and XylZ, are 201 residues shorter than the deduced BopZ sequence. As predicted from the sequence, expression of BopZ in E. coli yielded an approximately 60-kDa protein whose presence corresponded to increased cytochrome c reductase activity. While the N-terminal region of BopZ was approximately 50% identical in sequence to the entire BenC or XylZ reductases, the C terminus was unlike other known protein sequences.

Similarities between the antABC-Encoded Anthranilate Dioxygenase and the benABC-Encoded Benzoate Dioxygenase of Acinetobacter sp. Strain ADP1

ABSTRACT Acinetobacter sp. strain ADP1 can use benzoate or anthranilate as a sole carbon source. These structurally similar compounds are independently converted to catechol, allowing further degradation to proceed via the β-ketoadipate pathway. In this study, the first step in anthranilate catabolism was characterized. A mutant unable to grow on anthranilate, ACN26, was selected. The sequence of a wild-type DNA fragment that restored growth revealed theantABC genes, encoding 54-, 19-, and 39-kDa proteins, respectively. The deduced AntABC sequences were homologous to those of class IB multicomponent aromatic ring-dihydroxylating enzymes, including the dioxygenase that initiates benzoate catabolism. Expression of antABC in Escherichia coli, a bacterium that normally does not degrade anthranilate, enabled the conversion of anthranilate to catechol. Unlike benzoate dioxygenase (BenABC), anthranilate dioxygenase (AntABC) catalyzed catechol formation without requiring a dehydrogenase. In Acinetobacter mutants,benC substituted for antC during growth on anthranilate, suggesting relatively broad substrate specificity of the BenC reductase, which transfers electrons from NADH to the terminal oxygenase. In contrast, the benAB genes did not substitute for antAB. An antA point mutation in ACN26 prevented anthranilate degradation, and this mutation was independent of a mucK mutation in the same strain that prevented exogenous muconate degradation. Anthranilate induced expression of antA, although no associated transcriptional regulators were identified. Disruption of three open reading frames in the immediate vicinity ofantABC did not prevent the use of anthranilate as a sole carbon source. TheantABC genes were mapped on the ADP1 chromosome and were not linked to the two known supraoperonic gene clusters involved in aromatic compound degradation.