Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450

Creating artificial protein families affords new opportunities to explore the determinants of structure and biological function free from many of the constraints of natural selection. We have created an artificial family comprising ~3,000 P450 heme proteins that correctly fold and incorporate a heme cofactor by recombining three cytochromes P450 at seven crossover locations chosen to minimize structural disruption. Members of this protein family differ from any known sequence at an average of 72 and by as many as 109 amino acids. Most (>73%) of the properly folded chimeric P450 heme proteins are catalytically active peroxygenases; some are more thermostable than the parent proteins. A multiple sequence alignment of 955 chimeras, including both folded and not, is a valuable resource for sequence-structure-function studies. Logistic regression analysis of the multiple sequence alignment identifies key structural contributions to cytochrome P450 heme incorporation and peroxygenase activity and suggests possible structural differences between parents CYP102A1 and CYP102A2

Structure, dynamics, and function of the monooxygenase P450 BM-3: insights from computer simulations studies

The monooxygenase P450 BM-3 is a NADPH-dependent fatty acid hydroxylase enzyme isolated from soil bacterium Bacillus megaterium. As a pivotal member of cytochrome P450 superfamily, it has been intensely studied for the comprehension of structure-dynamics-function relationships in this class of enzymes. In addition, due to its peculiar properties, it is also a promising enzyme for biochemical and biomedical applications. However, despite the efforts, the full understanding of the enzyme structure and dynamics is not yet achieved. Computational studies, particularly molecular dynamics (MD) simulations, have importantly contributed to this endeavor by providing new insights at an atomic level regarding the correlations between structure, dynamics, and function of the protein. This topical review summarizes computational studies based on MD simulations of the cytochrome P450 BM-3 and gives an outlook on future directions

Unraveling Binding Effects of Cobalt(II) Sepulchrate with the Monooxygenase P450 BM-3 Heme Domain Using Molecular Dynamics Simulations

One of the major limitations to exploit enzymes in industrial processes is their dependence on expensive reduction equivalents like NADPH to drive their catalytic cycle. Soluble electron transfer (ET) mediators like Cobalt(II)Sepulchrate have been proposed as a cost-effective alternative to shuttle electrons between an inexpensive electron source and enzyme redox center. The interactions of these molecules with enzymes are not elucidated at molecular level yet. Herein, molecular dynamics simulations are performed to understand the binding and ET mechanism of the Cobalt(II)Sepulchrate with the heme domain of cytochrome P450BM-3. The study provides a detailed map of ET mediator binding sites on protein surface that resulted prevalently composed by Asp and Glu amino acids. The Cobalt(II)Sepulchrate do not show a preferential binding to these sites. However, among the observed binding sites, only few of them provide efficient ET pathways to heme iron. The results of this study can be used to improve the ET mediator efficiency of the enzyme for possible biotechnological applications

Dynamics and Interactions of Membrane Proteins

Membrane proteins are members of the class of proteins that perform their functions while being associated with a lipid bilayer. In the cell, they serve as transporters, receptors, anchors and enzymes. The domain organisation of these proteins suggests importance of lipid membrane and protein-lipid interactions for protein function. The requirement of a membrane mimic and the level of its resemblance to a native one for protein investigation makes the studies of membrane proteins a challenging project. My research work is focusing on the biophysical and biochemical studies of membrane proteins. This dissertation outlines two separate projects, each with their own challenges. Ras proteins are members of a superfamily of small GTPases that act as molecular switches that are involved in signal transduction pathways responsible for cell division and proliferation and, as one might guess, protein malfunction can lead to cancer. Recently, there have been a number of studies that suggest Ras protein dimerization on lipid membranes through protein-protein interactions between G- domains. On the basis of the results obtained from solution NMR and fluorescence polarization anisotropy studies, we concluded that the G-domain of the Ras protein by itself is not prone to dimerization. The result of this work was later confirmed by publications from other groups that performed studies in the presence of the lipid bilayer. NADPH-cytochrome P450 oxidoreductase (POR) is an integral membrane protein involved in an electron transport pathway transferring electrons from NADPH to cytochrome P450. The goal was achieved by application of lipid nanodisc technology, 13C-methyl extrinsic labeling coupled with Methyl-TROSY NMR technique that resulted in signals that showed differential sensitivity towards the redox state of the protein cofactors and conformational transitions of the protein. Moreover, results were obtained on a 600MHz instrument under protonated conditions. The goal of this project was the development of methodology to obtain structural data on a high-molecular weight protein associated with lipid nanodiscs in the presence of paramagnetic cofactors. Membrane proteins are challenging systems to research due to diverse interactions they experience on the membrane surface. In this dissertation I successfully utilized two approaches investigating this interactions: in my first project, I separately studied protein-protein interaction underlying the dimerization hypothesis, while in my second project I suggested the approach to explore conformational details and diverse interactions in a lipoprotein complex

Characterization of diverse natural variants of CYP102A1 found within a species of Bacillus megaterium

An extreme diversity of substrates and catalytic reactions of cytochrome P450 (P450) enzymes is considered to be the consequence of evolutionary adaptation driven by different metabolic or environmental demands. Here we report the presence of numerous natural variants of P450 BM3 (CYP102A1) within a species of Bacillus megaterium. Extensive amino acid substitutions (up to 5% of the total 1049 amino acid residues) were identified from the variants. Phylogenetic analyses suggest that this P450 gene evolve more rapidly than the rRNA gene locus. It was found that key catalytic residues in the substrate channel and active site are retained. Although there were no apparent variations in hydroxylation activity towards myristic acid (C14) and palmitic acid (C16), the hydroxylation rates of lauric acid (C12) by the variants varied in the range of >25-fold. Interestingly, catalytic activities of the variants are promiscuous towards non-natural substrates including human P450 substrates. It can be suggested that CYP102A1 variants can acquire new catalytic activities through site-specific mutations distal to the active site

Study of Electron Transfer through the Reductase Domain of Neuronal Nitric Oxide Synthase and Development of Bacterial Nitric Oxide Synthase Inhibitors

Crystal structure of neuronal Nitric Oxide Synthase reductase (nNOSr) implies that large-scale domain motion is essential for electron transfer. However, the details are not well understood. To address this, we generated a functioning “Cys-lite” version of nNOSr and then replaced the nNOSr Glu816 and Arg1229 residues with Cys in the FMN and FAD domains (CL5SS) in order to allow cross-domain disulfide bond formation under pH 9 or to cross-linking using bis-maleimides. Cross-linked CL5SS exhibited a =95% decrease in cytochrome c reductase activity and reduction of the disulfide bond restored the activities. The results demonstrate that a conformational equilibrium involving FMN domains motion is essential for the electron transfer. A graded lengthening of the bis-maleimide cross-linkers was associated with an increase in activity, thus helping to define the distance constraints for domain opening. Stopped-flow kinetic studies showed cross-linking did not negatively affect the hydride transfer and interflavin electron but severally impaired the electron efflux from the FMN domain to its redox partner. How these findings impact our understanding of the nNOS catalytic cycle and details are discussed. Staphylococcus aureus nitric oxide synthase (saNOS) helps S. aureus to maintain its antibiotics resistance, making saNOS a drug target. However, in vitro determination of saNOS inhibitor potency by activity assay is challenging because saNOS lacks an attached reductase. Herein, we employ the following approaches to optimize the in vitro assessment of NO synthesis by saNOS (1) B. subtillis flavodoxin YkuN and B. subtillis flavodoxin reductase FLDR were adopted as reductase partners for saNOS; (2) PEGylated-oxyhemoglobin was used for the direct capture of NO; (3) a 96-well plate format was used to increase the assay throughput. Our results showed that PEGylation of oxyHb minimizes the futile redox cycling within the flavoprotein and ensured effective electron transfer from produced NO to oxyHb. Nitric oxide produced by saNOS and cell cytosol was successfully detected by our assays. We also tested the inhibitory potency of six compounds derived from trimethoprim. They were confirmed to be H4F competitor with IC50 varying from 1 µM to 1 mM. The most potent inhibitor UCP111F26M is very specific to saNOS. Details of this inhibitor are discussed