Kinetic and structural studies of aldehyde oxidoreductase from Desulfovibrio gigas reveal a dithiolene-based chemistry for enzyme activation and inhibition by H(2)O(2).

Kinetic and structural studies of aldehyde oxidoreductase from Desulfovibrio gigas reveal a dithiolene-based chemistry for enzyme activation and inhibition by H(2)O(2).

Mononuclear Mo-containing enzymes of the xanthine oxidase (XO) household catalyze the oxidative hydroxylation of aldehydes and heterocyclic compounds. The molybdenum energetic website reveals a distorted square-pyramidal geometry by which two ligands, a hydroxyl/water molecule (the catalytic labile website) and a sulfido ligand, have been proven to be important for catalysis.

The XO member of the family aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is an exception as presents in its catalytically competent type an equatorial oxo ligand as an alternative of the sulfido ligand.

Kinetic and structural studies of aldehyde oxidoreductase from Desulfovibrio gigas reveal a dithiolene-based chemistry for enzyme activation and inhibition by H(2)O(2).
Kinetic and structural studies of aldehyde oxidoreductase from Desulfovibrio gigas reveal a dithiolene-based chemistry for enzyme activation and inhibition by H(2)O(2).

Despite this structural distinction, inactive samples of DgAOR could be activated upon incubation with dithionite plus sulfide, a process much like that used for activation of desulfo-XO. The proven fact that DgAOR doesn’t want a sulfido ligand for catalysis signifies that the method resulting in the activation of inactive DgAOR samples is totally different to that of desulfo-XO.

We now report a mixed kinetic and X-ray crystallographic examine to unveil the enzyme modification accountable for the inactivation and the chemistry that happens on the Mo website when DgAOR is activated.

In distinction to XO, which is activated by resulfuration of the Mo website, DgAOR activation/inactivation is ruled by the oxidation state of the dithiolene moiety of the pyranopterin cofactor, which demonstrates the non-innocent conduct of the pyranopterin in enzyme exercise.

We additionally confirmed that DgAOR incubation with dithionite plus sulfide within the presence of dioxygen produces hydrogen peroxide not related to the enzyme activation. The peroxide molecule coordinates to molybdenum in a η(2) vogue inhibiting the enzyme exercise.

Cloning, Baeyer-Villiger biooxidations, and constructions of the camphor pathway 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-coenzyme A monooxygenase of Pseudomonas putida ATCC 17453

A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate within the metabolism of camphor by Pseudomonas putida ATCC 17453, had been initially characterised in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W.

A comparability of a number of crystal kinds of OTEMO

Trudgill, J. Bacteriol. 153:140-152, 1983). Here we cloned and overexpressed the 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli and decided its three-dimensional construction with certain flavin adenine dinucleotide (FAD) at a 1.95-Å decision in addition to with certain FAD and NADP(+) at a 2.0-Å decision. OTEMO represents the primary homodimeric sort 1 BVMO construction certain to FAD/NADP(+).

A comparability of a number of crystal kinds of OTEMO certain to FAD and NADP(+) revealed a conformational plasticity of a number of loop areas, some of which have been implicated in contributing to the substrate specificity profile of structurally associated BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetic acid coenzyme.

A ester is most well-liked over the free acid. However, the catalytic effectivity (okay(cat)/Okay(m)) favors 2-n-hexyl cyclopentanone (4.3 × 10(5) M(-1) s(-1)) as a substrate, though its affinity (Okay(m) = 32 μM) was decrease than that of the CoA-activated substrate (Okay(m) = 18 μM).

whole-cell biotransformation experiments

In whole-cell biotransformation experiments, OTEMO confirmed a distinctive enantiocomplementarity to the motion of the prototypical cyclohexanone monooxygenase (CHMO) and seemed to be notably helpful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular construction and mechanistic complexity of the kind 1 household of BVMOs and expands the catalytic repertoire of one of its authentic members.

A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate within the metabolism of camphor by Pseudomonas putida ATCC 17453, had been initially characterised in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol.

153:140-152, 1983). Here we cloned and overexpressed the 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli and decided its three-dimensional construction with certain flavin adenine dinucleotide (FAD) at a 1.95-Å decision in addition to with certain FAD and NADP(+) at a 2.0-Å decision. OTEMO represents the primary homodimeric sort 1 BVMO construction certain to FAD/NADP(+).

A comparability of a number of crystal kinds of OTEMO certain to FAD and NADP(+) revealed a conformational plasticity of a number of loop areas, some of which have been implicated in contributing to the substrate specificity profile of structurally associated BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is most well-liked over the free acid. However, the catalytic effectivity (okay(cat)/Okay(m)) favors 2-n-hexyl cyclopentanone (4.3 × 10(5) M(-1) s(-1)) as a substrate, though its affinity (Okay(m) = 32 μM) was decrease than that of the CoA-activated substrate (Okay(m) = 18 μM).

In whole-cell biotransformation experiments, OTEMO confirmed a distinctive enantiocomplementarity to the motion of the prototypical cyclohexanone monooxygenase (CHMO) and seemed to be notably helpful for the oxidation of 4-substituted cyclohexanones.

Overall, this work extends our understanding of the molecular construction and mechanistic complexity of the kind 1 household of BVMOs and expands the catalytic repertoire of one of its authentic members.

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