Zulfiqar Hasan

Directed evolution was performed on vanadium chloroperoxidase from the fungus Curvularia inaequalis to increase its brominating activity at a mildly alkaline pH for industrial and synthetic applications and to further understand its mechanism. After successful expression of the enzyme in Escherichia coli, two rounds of screening and selection, saturation mutagenesis of a "hot spot," and rational recombination, a triple mutant (P395D/L241V/T343A) was obtained that showed a 100-fold increase in activity at pH 8 (k(cat) = 100 s(-1)). The increased K(m) values for Br(-) (3.1 mm) and H(2)O(2) (16 microm) are smaller than those found for vanadium bromoperoxidases that are reasonably active at this pH. In addition the brominating activity at pH 5 was increased by a factor of 6 (k(cat) = 575 s(-1)), and the chlorinating activity at pH 5 was increased by a factor of 2 (k(cat) = 36 s(-1)), yielding the "best" vanadium haloperoxidase known thus far. The mutations are in the first and second coordination sphere of the vanadate cofactor, and the catalytic effects suggest that fine tuning of residues Lys-353 and Phe-397, along with addition of negative charge or removal of positive charge near one of the vanadate oxygens, is very important. Lys-353 and Phe-397 were previously assigned to be essential in peroxide activation and halide binding. Analysis of the catalytic parameters of the mutant vanadium bromoperoxidase from the seaweed Ascophyllum nodosum also adds fuel to the discussion regarding factors governing the halide specificity of vanadium haloperoxidases. This study presents the first example of directed evolution of a vanadium enzyme.

We report 51V solid-state NMR spectroscopy of the 67.5-kDa vanadium chloroperoxidase, at 14.1 T. We demonstrate that, despite the low concentration of vanadium sites in the protein (one per molecule, 1 mumol of vanadium spins in the entire sample), the spinning sideband manifold spanning the central and the satellite transitions is readily detectable. The quadrupolar and chemical shift anisotropy tensors have been determined by numerical simulations of the spinning sideband envelopes and the line shapes of the individual spinning sidebands corresponding to the central transition. The observed quadrupolar coupling constant C(Q) of 10.5 +/- 1.5 MHz and chemical shift anisotropy delta(sigma) of -520 +/- 13 ppm are sensitive reporters of the geometric and electronic structure of the vanadium center. Density functional theory calculations of the NMR spectroscopic observables for an extensive series of active site models indicate that the vanadate cofactor is most likely anionic with one axial hydroxo- group and an equatorial plane consisting of one hydroxo- and two oxo- groups. The work reported in this manuscript is the first example of 51V solid-state NMR spectroscopy applied to probe the vanadium center in a protein directly. This approach yields the detailed coordination environment of the metal unavailable from other experimental measurements and is expected to be generally applicable for studies of diamagnetic vanadium sites in metalloproteins.

Nonspecific acid phosphatases share a conserved active site with mammalian glucose-6-phosphatases (G6Pase). In this work we examined the kinetics of the phosphorylation of glucose and dephosphorylation of glucose-6-phosphate (G6P) catalysed by the acid phosphatases from Shigella flexneri (PhoN-Sf) and Salmonella enterica (PhoN-Se). PhoN-Sf is able to phosphorylate glucose regiospecifically to G6P, glucose-1-phosphate is not formed. The K(m) for glucose using pyrophosphate (PPi) as a phosphate donor is 5.3 mM at pH 6.0. This value is not significantly affected by pH in the pH region 4-6. The K(m) value for G6P by contrast is much lower (0.02 mM). Our experiments show these bacterial acid phosphatases form a good model for G6Pase. We also studied the phosphorylation of inosine to inosine monophosphate (IMP) using PPi as the phosphate donor. PhoN-Sf regiospecifically phosphorylates inosine to inosine-5'-monophosphate whereas PhoN-Se produces both 5'IMP and 3'IMP. The data show that during catalysis an activated phospho-enzyme intermediate is formed that is able to transfer its phosphate group to water, glucose or inosine. A general mechanism is presented of the phosphorylation and dephosphorylation reaction catalysed by the acid phosphatases. Considering the nature of the substrates that are phosphorylated it is likely that this class of enzyme is able to phosphorylate a wide range of hydroxy compounds.