Molecular structure of D-hydantoinase from Bacillus sp. AR9: evidence for mercury inhibition.

Stereospecific conversion of hydantoins into their carbamoyl acid derivatives could be achieved by using the enzyme hydantoinase. Specific hydantoinases convert either the D-form or the L-form of the hydantoin and the amino acids responsible for stereospecificity have not been identified. Structural studies on hydantoinases from a few bacterial species were published recently. The structure of a thermostable D-hydantoinase from Bacillus sp. AR9 (bar9HYD) was solved to 2.3 angstroms resolution. The usual modification of carboxylation of the active-site residue Lys150 did not happen in bar9HYD. Two manganese ions were modelled in the active site. Through biochemical studies, it was shown that mercury inhibits the activity of the enzyme. The mercury derivative provided some information about the binding site of the mercuric inhibitors and a possible reason for inhibition is presented.

Serine protease inhibitor mediated peptide bond re-synthesis in diverse protein molecules.

Protease inhibitors have been extensively used in research to prevent unwanted degradation of proteins during purification and analysis. Here, we report a remarkable discovery of protease inhibitor mediated reformation of peptide bonds by the serine protease inhibitor, PMSF in a diverse set of proteolyzed molecules. Interestingly, the religation reaction in the presence of PMSF occurs in a very short time period and with very high yields of the religated product. We also investigate the plausible mechanism of such a reaction and demonstrate through biochemical studies and X-ray crystallography that proximity of reacting termini is essential for the feasibility of this reaction.

Carboxylated lysine is required for higher activities in Hydantoinases.

Hydantoinases are industrial enzymes with varying degree of activities on variable substrates to form different products. Although, few of the hydantoinase structures were known recently, the functional details and active site mechanism were not clearly understood yet. In a structure determination effort we reported that Bacillus sp. AR9 hydantoinase contains uncarboxylated lysine in the active site, whereas all the other hydantoinases have a carboxylated active site lysine. Here we describe the importance of carboxylated lysine for differential activities by making lysine mutations as well as carboxylating the lysine in a D-hydantoinase from Bacillus sp. AR9. The lysine to alanine and lysine to arginine mutations showed reduced activities whereas carboxylation of the lysine has enhanced the activity. Theoretical studies involving the calculation of electrostatic potentials for the hydroxide ion between the two metal ions present in the active site suggest that the presence of carboxylated lysine increases the nucleophilicity of the hydroxide.

Characterization of Mycobacterium tuberculosis WhiB1/Rv3219 as a protein disulfide reductase.

WhiB family of protein is emerging as one of the most fascinating group and is implicated in stress response as well as pathogenesis via their involvement in diverse cellular processes. Surprisingly, available in vivo data indicate an organism specific physiological role for each of these proteins. The WhiB proteins have four conserved cysteine residues where two of them are present in a C-X-X-C motif. In thioredoxins and similar proteins, this motif works as an active site and confers thiol-disulfide oxidoreductase activity to the protein. The recombinant WhiB1/Rv3219 was purified in a single step from Escherichia coli using Ni(2+)-NTA affinity chromatography and was found to exist as a homodimer. Mass spectrometry of WhiB1 shows that the four cysteine residues form two intramolecular disulfide bonds. Using intrinsic tryptophan fluorescence as a measure of redox state, the redox potential of WhiB1 was calculated as -236+/-2mV, which corresponds to the redox potential of many cytoplasmic thioredoxin-like proteins. WhiB1 catalyzed the reduction of insulin disulfide thus clearly demonstrating that it functions as a protein disulfide reductase. Present study for the first time suggests that WhiB1 may be a part of the redox network of Mycobacterium tuberculosis through its involvement in thiol-disulfide exchange with other cellular proteins.

A novel UV laser-induced visible blue radiation from protein crystals and aggregates: scattering artifacts or fluorescence transitions of peptide electrons delocalized through hydrogen bonding?

Proteins lacking prosthetic groups and/or cofactors are known to undergo electronic excitation transitions only upon exposure to UV-C (< 280 nm) and UV-B (280-320 nm), but not UV-A (320-400 nm) photons. Here, we report the discovery of a novel excitation that peaks at approximately 340 nm and yields visible violet-blue radiation with apparent band maxima at approximately 425, 445, 470, and 500 nm. All proteins and large polypeptides examined in solid form, and in solutions, display this quenchable and photobleachable radiation which can be established not owing to aromatic sidechains. As a note of caution, we wish to state that we have not been able to completely eliminate the possibility that the radiation may be an artifact owing to second order effects such as, e.g., Raman scattering of Raman-scattered photons; however, we assert that all our experiments indicate that the radiation actually owes to some form of fluorescence. We propose that peptide electrons that have been delocalized through intramolecular or intermolecular hydrogen bond formation display these long-wavelength electronic transitions. If confirmed by future studies, this preliminary discovery may turn out to have important implications for biomolecular spectroscopy, protein crystallography, and materials science.