Characterization of the N-oxygenase AurF from Streptomyces thioletus.

AurF catalyzes the N-oxidation of p-aminobenzoic acid to p-nitrobenzoic acid in the biosynthesis of the antibiotic aureothin. Here we report the characterization of AurF under optimized conditions to explore its potential use in biocatalysis. The pH optimum of the enzyme was established to be 5.5 using phenazine methosulfate (PMS)/NADH as the enzyme mediator system, showing ~10-fold higher activity than previous reports in literature. Kinetic characterization at optimized conditions give a Km of 14.7 ± 1.1 µM, a kcat of 47.5 ± 5.4 min(-1) and a kcat/Km of 3.2 ± 0.4 µM(-1)min(-1). PMS/NADH and the native electron transfer proteins showed significant formation of the p-hydroxylaminobenzoic acid intermediate, however H2O2 produced mostly p-nitrobenzoic acid. Alanine scanning identified the role of important active site residues. The substrate specificity of AurF was examined and rationalized based on the protein crystal structure. Kinetic studies indicate that the Km is the main determinant of AurF activity toward alternative substrates.

CmlI is an N-oxygenase in the biosynthesis of chloramphenicol.

The N-oxygenation of an amine group is one of the steps in the biosynthesis of the antibiotic chloramphenicol. The non-heme di-iron enzyme CmlI was identified as the enzyme catalyzing this reaction through bioinformatics studies and reconstitution of enzymatic activity. In vitro reconstitution was achieved using phenazine methosulfate and NADH as electron mediators, while in vivo activity was demonstrated in Escherichia coli using two substrates. Kinetic analysis showed a biphasic behavior of the enzyme. Oxidized hydroxylamine and nitroso compounds in the reaction were detected both in vitro and in vivo based on LC-MS. The active site metal was confirmed to be iron based on a ferrozine assay. These findings provide new insights into the biosynthesis of chloramphenicol and could lead to further development of CmlI as a useful biocatalyst.

Quantitative analysis of Poisson-Boltzmann implicit solvent in molecular dynamics.

A critical issue in the development of implicit solvent models is their quality in realistic simulations of non-trivial systems. In a previous study, we quantitatively compared the reaction field energies of static structures calculated with the Poisson-Boltzmann implicit solvent and the TIP3P explicit solvent and found an overall agreement, though a discrepancy was also observed in the electrostatic potentials of mean force for salt-bridging and hydrogen-bonding dimers (see J. Phys. Chem. B, 2006, 110, 18680). In this study, we are interested in how the implicit solvent performs in molecular dynamics simulations. To guarantee sampling convergence in simulated observables in the explicit solvent, we explored to use a high-temperature constant-volume simulation setting at 450 K but with the water density at 300 K. The relevance of the artificial simulation setting to room-temperature simulations of biomolecules was first investigated by systematic comparisons of the polar and nonpolar solvation free energies of 23 amino acid analogues at 300 K and 450 K, respectively. Assisted by the artificial simulation setting, we found the simulated secondary structure populations agree very well between the implicit and explicit solvents for tested dipeptides and peptides. In addition, the agreement in the populations of hydrophobic contacts is reasonable. However, our analysis also shows that the populations of the salt bridges are too low in the implicit solvent. The low salt-bridge population perhaps results from a combination of the atomic-centered modified van der Waals surface and the small solvent probe radius optimized to best reproduce the polar potential of mean force profiles. In addition, the lower accuracy of the electrostatic forces and the lack of water-bridged minima in the implicit solvents may also contribute to the instability of the salt bridge populations.