4-Nitrobenzyl alcohol

Pseudomonas sp. strain TW3 is able to metabolize 4-nitrotoluene to 4-nitrobenzoate and toluene to benzoate aerobically via a route analogous to the upper pathway of the TOL plasmids. We report the cloning and characterization of a benzyl alcohol dehydrogenase gene (ntnD) which encodes the enzyme for the catabolism of 4-nitrobenzyl alcohol and benzyl alcohol to 4-nitrobenzaldehyde and benzaldehyde, respectively. The gene is located downstream of the previously reported ntn gene cluster. NtnD bears no similarity to the analogous TOL plasmid XylB (benzyl alcohol dehydrogenase) protein either in its biochemistry, being NAD(P)(+) independent and requiring assay via dye-linked electron transfer, or in its deduced amino acid sequence. It does, however, have significant similarity in its amino acid sequence to other NAD(P)(+)-independent alcohol dehydrogenases and contains signature patterns characteristic of type III flavin adenine dinucleotide-dependent alcohol oxidases. Reverse transcription-PCR demonstrated that ntnD is transcribed during growth on 4-nitrotoluene, although apparently not as part of the same transcript as the other ntn genes. The substrate specificity of the enzyme expressed from the cloned and overexpressed gene was similar to the activity expressed from strain TW3 grown on 4-nitrotoluene, providing evidence that ntnD is the previously unidentified gene in the pathway of 4-nitrotoluene catabolism. Examination of the 14.8-kb region around the ntn genes suggests that one or more recombination events have been involved in the formation of their current organization.

The compounds 3-hydroxy-4-nitrobenzaldehyde and 3-hydroxy-4-nitrobenzyl alcohol are introduced as new chromophoric substrates for probing the catalytic mechanism of horse liver alcohol dehydrogenase (LADH). Ionization of the phenolic hydroxyl group shifts the spectrum of the aldehyde from 360 to 433 nm (pKa = 6.0), whereas the spectrum of the alcohol shifts from 350 to 417 nm (pKa = 6.9). Rapid-scanning, stopped-flow (RSSF) studies at alkaline pH show that the LADH-catalyzed interconversion of these compounds occurs via the formation of an enzyme-bound intermediate with a blue-shifted spectrum. When reaction is limited to a single turnover of enzyme sites, the formation and decay of the intermediate when aldehyde reacts with enzyme-bound reduced nicotinamide adenine dinucleotide E(NADH) are characterized by two relaxations (lambda f approximately equal to 3 lambda s). Detailed stopped-flow kinetic studies were carried out to investigate the disappearance of aldehyde and NADH, the formation and decay of the intermediate, the displacement of Auramine O by substrate, and 2H kinetic isotope effects. It was found that NADH oxidation takes place at the rate of the slower relaxation (lambda s); when NADD is substituted for NADH, lambda s is subject to a small primary isotope effect (lambda Hs/lambda Ds = 2.0); and the events that occur in lambda s precede lambda f. These findings identify the intermediate as a ternary complex containing bound oxidized nicotinamide adenine dinucleotide (NAD+) and some form of 3-hydroxy-4-nitrobenzyl alcohol. The blue-shifted spectrum of the intermediate strongly implies a structure wherein the phenolic hydroxyl is neutral. When constrained to a mechanism that assumes only the neutral phenolic form of the substrate binds and reacts and that the intermediate is an E(NAD+, product) complex, computer simulations yield RSSF and single-wavelength time courses that are qualitatively and semiquantitatively consistent with the experimental data. We conclude that the LADH substrate site can be divided into two subsites: a highly polar, electropositive subsite in the vicinity of the active-site zinc and, just a few angstroms away, a rather nonpolar region. The polar subsite promotes formation of the two interconverting reactive ternary complexes. The nonpolar region is the binding site for the hydrocarbon-like side chains of substrates and in the case of 3-hydroxy-4-nitrobenzaldehyde conveys specificity for the neutral form of the phenolic group.