Identification and characterization of a novel (-)-vibo-quercitol 1-dehydrogenase from Burkholderia terrae suitable for production of (-)-vibo-quercitol from 2-deoxy-scyllo-inosose.

(-)-vibo-Quercitol is a deoxyinositol (1L-1,2,4/3,5-cyclohexanepentol) that naturally occurs in oak species, honeydew honey, wines aged in oak barrels, and Gymnema sylvestre and is a potential intermediate for pharmaceuticals. We found that (-)-vibo-quercitol is stereoselectively synthesized from 2-deoxy-scyllo-inosose by the reductive reaction of a novel (-)-vibo-quercitol 1-dehydrogenase found in the proteomes of Burkholderia, Pseudomonas, and Arthrobacter. Among them, Burkholderia terrae sp. AKC-020 (40-1) produced a (-)-vibo-quercitol 1-dehydrogenase appropriate for synthesizing (-)-vibo-quercitol with a high diastereomeric excess. The enzyme was strongly induced in Bu. terrae cells when quercitol or 2-deoxy-scyllo-inosose was used as carbon source in the culture medium. The enzyme is NAD(H)-dependent and shows highly specific activity for (-)-vibo-quercitol and myo-inositol among the substrates tested. The enzyme gene (qudh) was obtained by PCR using degenerate primers based on the N-terminal and internal amino acid sequences of the purified enzyme, followed by thermal asymmetric interlaced PCR. The qudh gene showed homology with inositol 2-dehydrogenase (sharing 49.5% amino acid identity with IdhA from Sinorhizobium meliloti 1021). We successfully produced several recombinant (-)-vibo-quercitol 1-dehydrogenases and related enzymes identified by genome database mining using an Escherichia coli expression system. This revealed that scyllo-inositol dehydrogenase (IolX) in Bacillus subtilis can catalyze the reduction of 2-deoxy-scyllo-inosose to yield scyllo-quercitol, a stereoisomer of (-)-vibo-quercitol. Thus, we successfully identified two enzymes to produce both stereoisomers of deoxyinositols that are rare in nature.

Discovery of a reaction intermediate of aliphatic aldoxime dehydratase involving heme as an active center.

Recently, we discovered an intriguing hemoprotein [aliphatic aldoxime dehydratase (OxdA)] that catalyzes the dehydration of aliphatic aldoximes [R-CH=N-OH] to the corresponding nitriles [R-C identical withN] in the industrial Pseudomonas chlororaphis B23 strain. Unlike the utilization of H(2)O(2) or O(2) as a mediator of the catalysis by other heme-containing enzymes (e.g., P450), OxdA is notable for the direct binding of a substrate to the heme iron, experimental evidence of which was obtained here by means of resonance Raman (RR) analysis with an isotope technique. We found that the addition of a large amount of butyraldoxime (final concentration, 200 mM) to ferrous OxdA with a low enzyme concentration (final concentration, 5 muM) yields a long-lived OxdA-substrate complex (named OS-II), whose UV-vis spectrum is different from the corresponding spectra of the OxdA-substrate complex I and CO-bound, ferrous, and ferric forms of OxdA. Intriguingly, the RR analysis demonstrated that OS-II includes a highly oxidized heme with strong bonding between a substrate and the heme iron, as judged from the heme oxidation state marker nu(4) band at 1,379 cm(-1) and the (15)N-isotope-substituted butyraldoxime sensitive band at 857 cm(-1) in the RR spectra. It is noteworthy that OS-II has a highly oxidized heme like the ferryl-oxo heme species (e.g., compound II) formed by some general hemoproteins, although the function of OxdA is different from those (transport of electrons, transport of oxygen, sensing of oxygen or carbon monoxide, and catalysis of redox reactions) of general hemoproteins.

Stopped-flow spectrophotometric and resonance Raman analyses of aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis.

On stopped-flow analysis of aliphatic aldoxime dehydratase (OxdA), a novel hemoprotein, a spectrum derived from a reaction intermediate was detected on mixing ferrous OxdA with butyraldoxime; it gradually changed into that of ferrous OxdA with an isosbestic point at 421 nm. The spectral change on the addition of butyraldoxime to the ferrous H320A mutant showed the formation of a substrate-coordinated mutant, the absorption spectrum of which closely resembled that of the above intermediate. These observations and the resonance Raman investigation revealed that the substrate actually binds to the heme in OxdA, forming a hexa-coordinate low-spin heme.

Identification of crucial histidines involved in carbon-nitrogen triple bond synthesis by aldoxime dehydratase.

Aldoxime dehydratase (OxdA), which is a novel heme protein, catalyzes the dehydration of an aldoxime to a nitrile even in the presence of water in the reaction mixture. The combination of site-directed mutagenesis of OxdA (mutation of all conserved histidines in the aldoxime dehydratase superfamily), estimation of the heme contents and specific activities of the mutants, and CD and resonance Raman spectroscopic analyses led to the identification of the proximal and distal histidines in this unique enzyme. The heme contents and CD spectra in the far-UV region of all mutants except for the H299A one were almost identical to those of the wild-type OxdA, whereas the H299A mutant lost the ability of binding heme, demonstrating that His(299) is the proximal histidine. On the other hand, substitution of alanine for His(320) did not affect the overall structure of OxdA but caused loss of its ability of carbon-nitrogen triple bond synthesis and a lower shift of the Fe-C stretching band in the resonance Raman spectrum for the CO-bound form. Furthermore, the pH dependence of the wild-type OxdA closely followed the His protonation curves observed for other proteins. These findings suggest that His(320) is located in the distal heme pocket of OxdA and would donate a proton to the substrate in the aldoxime dehydration mechanism.

Heme environment in aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis.

Resonance Raman spectra have been measured to characterize the heme environment in aldoxime dehydratase (OxdA), a novel hemoprotein, which catalyzes the dehydration of aldoxime into nitrile. The spectra showed that the ferric heme in the enzyme is six-coordinate low spin, whereas the ferrous heme is five-coordinate high spin. We assign a prominent vibration that occurs at 226 cm(-1) in the ferrous enzyme to the Fe-proximal histidine stretching vibration. In the CO-bound form of OxdA, the correlation between the Fe-CO stretching (512 cm(-1)) and C-O stretching (1950 cm(-1)) frequencies also supports our assignment of proximal histidine coordination.

Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas chlororaphis B23. Sequencing, gene expression, purification, and characterization.

Analysis of the nitrile hydratase gene cluster involved in nitrile metabolism of Pseudomonas chlororaphis B23 revealed that it contains one open reading frame encoding aldoxime dehydratase upstream of the amidase gene. The amino acid sequence deduced from this open reading frame shows similarity (32% identity) with that of Bacillus phenylacetaldoxime dehydratase (Kato, Y., Nakamura, K., Sakiyama, H., Mayhew, S. G., and Asano, Y. (2000) Biochemistry 39, 800-809). The gene product expressed in Escherichia coli catalyzed the dehydration of aldoxime into nitrile. The Pseudomonas aldoxime dehydratase (OxdA) was purified from the E. coli transformant and characterized. OxdA shows an absorption spectrum with a Soret peak that is characteristic of heme, demonstrating that it is a hemoprotein. For its activity, this enzyme required a reducing reagent, Na2S2O4, but did not require FMN, which is crucial for the Bacillus enzyme. The enzymatic reaction was found to be catalyzed when the heme iron of the enzyme was in the ferrous state. Calcium as well as iron was included in the enzyme. OxdA reduced by Na2S2O4 had a molecular mass of 76.2 kDa and consisted of two identical subunits. The kinetic parameters of OxdA indicated that aliphatic aldoximes are more effective substrates than aromatic aldoximes. A variety of spectral shifts in the absorption spectra of OxdA were observed upon the addition of each of various compounds (i.e. redox reagents and heme ligands). Moreover, the addition of the substrate to OxdA gave a peak that would be derived from the intermediate in the nitrile synthetic reaction. P. chlororaphis B23 grew and showed the OxdA activity when cultured in a medium containing aldoxime as the sole carbon and nitrogen source. Together with these findings, Western blotting analysis of the extracts using anti-OxdA antiserum revealed that OxdA is responsible for the metabolism of aldoxime in vivo in this strain.