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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 A 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 >105 M−1 s−1 at pH 8.5 and 25 °C. Hammett plots for Km and kcat using substituted salicylates indicate 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.

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.

Polycyclic aromatic hydrocarbons (PAHs) are a group of aromatic hydrocarbons composed of two or more fused rings and include naphthalene, phenanthrene, pyrene and benzo[a]pyrene amongst others. Exposure to naphthalene has been associated with several toxic manifestations in humans and laboratory animals, with the lens of the eye and the lungs being most sensitive [1]. Additionally, this compound has been reclassified as a possible human carcinogen by the US Environmental Protection Agency and the International Agency for Research on Cancer. It has recently been suggested that naphthalene undergoes metabolic activation to 1,2-naphthoquinone, which reacts with DNA, leading to the formation of apurinic sites on DNA and depurinating DNA adducts [2]. Moreover, a number of studies have attested to the carcinogenic and mutagenic properties of more complex PAHs, suggesting the need for further work on the elimination of these compounds from the environment. The Gram-negative bacterium P. putida G7 is among the best studied naphthalene-degrading species. The genes associated with naphthalene metabolism are localized on NAH7, an 83 kilobase plasmid. In P. putida G7 the naphthalene-oxidation genes are organized into two operons under salicylate control. The first operon (upper naphthalene-degradation pathway) includes the genes nahAaAbAcAdBCDEF, which code for the conversion of naphthalene to salicylate, while the second operon (lower pathway) includes the genes nahGTHINLOMKJ responsible for the oxidation of salicylate via the catechol meta-cleavage pathway [3]. In the last years, our group focused on the structure elucidation and the kinetic characterization of the P. putida G7 enzymes involved in the naphthalene degradation pathway. We intend to present these results which basically describe the 3D structures of NahB, NahF, NahG, NahI, NahK and NahK/NahL complex and some kinetic data of these enzymes and their mutants. This work was supported by FAPEMIG, CNPq and VALE S.A.