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.

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.

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.

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.

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.