No abstract

This work provides functional data showing that the bacterial CYP102A1 recognises compounds metabolised by human CYP3A4, CYP2E1 and CYP1A2 and is able to catalyse different reactions. Wild-type cytochrome CYP102A1 from Bacillus megaterium is a catalytically self-sufficient enzyme, containing an NADPH-dependent reductase and a P450 haem domain fused in a single polypeptidie chain. An NADPH-dependent method (Tsotsou et al. in Biosens. Bioelectron. 17:119-131, 2002) together with spectroscopic assays were applied to investigate the catalytic activity of CYP102A1 towards 19 xenobiotics, including 17 commercial drugs. These molecules were chosen to represent typical substrates of the five main families of drug-metabolising human cytochromes P450. Liquid chromatography-mass spectrometry analysis showed that CYP102A1 catalyses the hydroxylation of chlorzoxazone, aniline and p-nitrophenol, as well as the N-dealkylation of propranolol and the dehydrogenation of nifedipine. These drugs are typical substrates of human CYP2E1 and CYP3A4. The KM values calculated for these compounds were in the millimolar range: 1.21+/-0.07 mM for chlorzoxazone, 2.52 +/- 0.08 mM for aniline, 0.81+/-0.04 mM for propranolol. The values of vmax for chlorzoxazone and propranolol were 46.0+/-9.0 and 7.6+/-3.4 nmol min-1 nmol-1, respectively. These values are higher then those measured for the human enzymes. The vmax value for aniline was 9.4+/-1.3 nmol min-1 nmol-1, comparable to that calculated for human cytochromes P450. The functional data were found to be in line with the sequence alignments, showing that the identity percentage of CYP102A1 with CYP3A4 and CYP2E1 is higher than that found for CYP1A2, CYP2C9 and CYP2D6 families.

In this work the catalytic properties of a cytochrome P450 immobilised onto an electrode surface are improved by means of the molecular Lego approach.

The crystallographic structure of an engineered flavodoxin mutant from Desulfovibrio vulgaris has been analysed. Site-directed mutagenesis was used to substitute serine 35 with a cysteine to provide a possible covalent linkage. The crystal structure of the semiquinone form of this mutant is similar to the corresponding oxidation state of the wild-type flavodoxin. Analysis of the structural changes reveals the interaction between N(5)H of the flavin and the carbonyl O atom of Gly61 to be critical for modulation of the electrochemical properties of the protein.

The crystal structures of the monomer and homodimer of S64C mutant from D. vulgaris in the oxidized and semiquinone states have been determined, at 1.80Å resolution with diffraction data at 100K, by molecular replacement using as starting model the wild type structure [1]. The interest for the new mutant S64C is due to the location of Cys64 in a key region for the interaction with the cofactor FMN and in a position favourable to the formation of homo-and heterodimers.The structures of ox and sq S64C monomer crystallizes in the space group P4 3 2 1 2 and have the general fold of flavodoxin family. The pattern of hydrogen bonds between the protein and FMN is similar to that of the wild type. The main structural differences between the ox and sq monomer are in the loop-60, that is involved in a new hydrogen bond with the cofactor upon reduction [2]. The two forms of the dimeric S64C mutant crystallize in the space group P4 1 2 1 2 and the dimer link is due to Cys64, that makes the disulfide bridge with a simmetry related mate. These structural studies provide the seminal information towards a better understanding of the role of the protein moiety in tuning the redox potential and, therefore, the electron transfer. The inhibitory loop of a serine protease inhibitor has a characteristic conformation while the remaining part of the molecule, known as scaffold, has widely different folds in different families of inhibitors. To understand the exact contribution of the 'inhibitor scaffold' towards the inhibition process, a classic -trefoil fold protein, Winged bean Chymotrypsin Inhibitor (WCI), has been chosen. Owing to the crucial strategic location, as seen in our previous crystallographic and molecular dynamics studies, a scaffolding residue Asn14 has been targeted for mutagenesis by residues of different shapes and charges and the ability of chymotrypsin inhibition by the resulting mutants has been measured. Crystal structures of the mutants were determined and it was observed that the degree of loop deformation is inversely proportional to the extent of chymotrypsin inhibition.Similarly, through mutations in the WCI loop region, two chimeric proteins are attempted with loops of trypsin inhibitors like ETI and STI on the scaffold of WCI. A comparison of binding constants of these chimeric proteins with their respective wild type ones can be used to understand whether the scaffold of WCI is best suited for chymotrypsin inhibition or it can be used for trypsin inhibition as well. As a first step towards this approach, we found that the single mutation (Leu 65 Arg) at P1 converts WCI to a potent inhibitor of trypsin with a K i value comparable to ETI and STI indicating that the role of the scaffold of WCI is comparable to that of ETI and STI. Structure of this mutant (L65R) PvuII is the first restriction endonuclease which has been converted from its wild-type (wt) homodimeric form into a single chain (sc) protein by tandemly joining the two subunits through the peptide linker GlySerGlyGly. The DNA cleavage activity of...