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By a targeted enzyme engineering approach, we were able to create an efficient NADPH oxidase from a monooxygenase. Intriguingly, replacement of only one specific single amino acid was sufficient for such a monooxygenase-to-oxidase switch--a complete transition in enzyme activity. Pre-steady-state kinetic analysis and elucidation of the crystal structure of the C65D PAMO mutant revealed that the mutation introduces small changes near the flavin cofactor, resulting in a rapid decay of the peroxyflavin intermediate. The engineered biocatalyst was shown to be a thermostable, solvent tolerant, and effective cofactor-regenerating biocatalyst. Therefore, it represents a valuable new biocatalytic tool. [ABSTRACT FROM AUTHOR]

Thymidylate is a DNA nucleotide that is essential to all organisms and is synthesized by the enzyme thymidylate synthase (TSase). Several human pathogens rely on an alternative flavin-dependent thymidylate synthase (FDTS), which differs from the human TSase both in structure and molecular mechanism. It has recently been shown that FDTS catalysis does not rely on an enzymatic nucleophile and that the proposed reaction intermediates are not covalently bound to the enzyme during catalysis, an important distinction from the human TSase. Here we report the chemical trapping, isolation, and identification of a derivative of such an intermediate in the FDTS-catalyzed reaction. The chemically modified reaction intermediate is consistent with currently proposed FDTS mechanisms that do not involve an enzymatic nucleophile, and it has never been observed during any other TSase reaction. These findings establish the timing of the methylene transfer during FDTS catalysis. The presented methodology provides an important experimental tool for further studies of FDTS, which may assist efforts directed toward the rational design of inhibitors as leads for future antibiotics. [ABSTRACT FROM AUTHOR]

Flavins are central to the reactivity of a wide variety of enzymes and electron transport proteins. There is great interest in understanding the basis for the different reactivities displayed by flavins in different protein contexts. We propose solid-state nuclear magnetic resonance (SS-NMR) as a tool for directly observing reactive positions of the flavin ring and thereby obtaining information on their frontier orbitals. We now report the SS-NMR signals of the redox-active nitrogens Ni and N5, as well as that of N3. The chemical shift tensor of N5 is over 720 ppm wide, in accordance with the predictions of theory and our calculations. The signal of N3 can be distinguished on the basis of coupling to ±H absent for Ni and N5, as well as the shift tensor span of only 170 ppm, consistent with N3's lower aromaticity and lack of a nonbonding lone pair. The isotropic shifts and spans of N5 and Ni reflect two opposite extremes of the chemical shift range for ‘pyridine-type’ N's, consistent with their electrophilic and nucleophilic chemical reactivities, respectively. Upon flavin reduction, N5's chemical shift tensor contracts dramatically to a span of less than 110 ppm, and the isotropic chemical shift changes by approximately 300 ppm. Both are consistent with loss of N5's nonbonding lone pair and decreased aromaticity, and illustrate the responsiveness of the 15N chemical shift principal values to electronic structure. Thus. 15N chemical shift principal values promise to be valuable tools for understanding electronic differences that underlie variations in flavin reactivity, as well as the reactivities of other heterocyclic cofactors. [ABSTRACT FROM AUTHOR]

The article examines the origin of rate enhancement of flavin mono-nucleotide (FMN). It states that FMN reduction can be monitored as a pseudo-first-order reaction using single-turnover approaches. It suggests that the difference in observed rate between two near identical coenzymes is related to a vibrational mode present in the pentaerythritol tetranitrate reductase (PETNR) NADPH complex.

The article discusses the participation of flavin in product formation by thyX-encoded thymidylate synthase (TS). Thymine for DNA is synthesized by the enzyme TS. Many pathogenic bacteria lack the well-known TS encoded by the thyA gene and instead synthesize thymidylate (dTMP) with a TS encoded by the thyX gene. The likely difference in mechanism from thyA-encoded TS and its occurrence in many pathogenic bacteria make thyX-encoded TS a promising drug target. The article reports studies on the oxidative hall-reaction of the thyX gene product from Campylobacter jejuni that establish the stoichiometry of conversion of 2'-deoxyuridine monophosphate and 5,10-methylene- tetrahydrofolate into dTMP and tetrahydrofolate.