Salicylate hydroxylase (NahG) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of salicylate into catechol in the naphthalene degradation pathway in Pseudomonas putida G7. We explored the mechanism of action of this enzyme in detail using a combination of structural and biophysical methods. NahG shares many structural and mechanistic features with other versatile flavin-dependent monooxygenases, with potential biocatalytic applications. The crystal structure at 2.0 Å resolution for the apo form of NahG adds a new snapshot preceding the FAD binding in flavin-dependent monooxygenases. The kcat /Km for the salicylate reaction catalyzed by the holo form is greater than 10 5 M -1 s -1 at pH 8.5 and 25 ºC.Hammett plots for Km and kcat using substituted salicylates indicates a change in rate-limiting step.Electron-donating groups favor the hydroxylation of salicylate by a peroxyflavin to yield a Wheland-like intermediate, whereas the decarboxylation of this intermediate is faster for electron-withdrawing groups. The mechanism is supported by structural data and kinetic studies at different pHs. The salicylate carboxyl group lies near a hydrophobic region that aids decarboxylation. A conserved histidine residue is proposed to assist the reaction by general base/general acid catalysis.to the substrate phenol group [22]. In the reaction catalyzed by 3-hydroxybenzoate 6hydroxylase (3HB6H), which the substrate carboxylate group is meta to the phenol, the 3-hydroxybenzoate is converted to 2,5-dihydroxybenzoate through a hydroxylation followed by a deprotonation [23].Catalysis in these enzymes involves a C(4a)-hydroperoxyflavin species, which provides a powerful peroxo electrophile tuned for oxygen insertion at nucleophilic carbons and soft centers.Examples include hydroxylation coupled with different kinds of substitution, epoxidation, Baeyer-Villiger oxidation, and oxidation of heteroatoms (B, S, Se, N, and P) [24][25][26][27]. Formation of the C(4a)-hydroperoxyflavin species from the oxidized flavin and NAD(P)H requires two reactions coupled with dynamics of the isoalloxazine group between two positions [28]. In an external position, aside from the hydroxylation site, the oxidized isoaloxazine group is reduced by NAD(P)H. Then, the reduced isoaloxazine swings to an internal position, where it reacts with molecular oxygen to yield the C(4a)-hydroperoxyflavin species. This reaction is fast in enzymes, with rate constants typically between 10 4 and 10 6 M -1 s -1 at 4 ºC [29][30][31][32]; in contrast, the rate constant for the corresponding reaction in water is about 250 M -1 s -1 at 30 ºC [33].Herein, we explore the catalytic mechanism of NahG, that, like those other monooxygenases, remains a subject of intense debate [10,20]. Particular goals are to identify the catalytic groups involved in catalysis, the reactivity of reaction intermediates, and the reaction pathway that affords the products. The mechanistic proposal resulting from this work is supported by kinetics and x-ray crystallogra...

The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD(+) as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 10(6) M(-1) s(-1). The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes.

Phosphoimidazole-containing compounds are versatile players in biological and chemical processes. We explore catalytic and mechanistic criteria for the efficient formation of cyclic aryl phosphoimidazoles in aqueous solution, viewed as a template reaction for the in situ synthesis of related compounds. To provide a detailed analysis for this reaction a series of o-(2'-imidazolyl)naphthyl (4-nitrophenyl) phosphate isomers were examined to provide a basis for analysis of both mechanism and the influence of structural factors affecting the nucleophilic attack of the imidazolyl group on the phosphorus center of the substrate. Formation of the cyclic aryl phosphoimidazoles was probed by NMR and ESI-MS techniques. Kinetic experiments show that cyclization is faster under alkaline conditions, with an effective molarity up to 2900 M for the imidazolyl group, ruling out competition from external nucleophiles. Heavy atom isotope effect and computational studies show that the reaction occurs through a S2(P)-type mechanism involving a pentacoordinated phosphorus TS, with apical positions occupied by the incoming imidazolyl nucleophile and the p-nitrophenolate leaving group. The P-O bond to the leaving group is about 50-60% broken in the transition state.

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