Membrane-bound P450s Research Articles

The CYP79A1 catalyzed conversion of tyrosine to (E)-p-hydroxyphenylacetaldoxime unravelled using an improved method for homology modeling

The vast diversity and membrane-bound nature of plant P450s makes it challenging to study the structural characteristics of this class of enzymes especially with respect to accurate intermolecular enzyme-substrate interactions. To address this problem we here apply a modified hybrid structure strategy for homology modeling of plant P450s. This allows for structural elucidation based on conserved motifs in the protein sequence and secondary structure predictions. We modeled the well-studied Sorghum bicolor cytochrome P450 CYP79A1 catalyzing the first step in the biosynthesis of the cyanogenic glucoside dhurrin. Docking experiments identified key regions of the active site involved in binding of the substrate and facilitating catalysis. Arginine 152 and threonine 534 were identified as key residues interacting with the substrate. The model was validated experimentally using site-directed mutagenesis. The new CYP79A1 model provides detailed insights into the mechanism of the initial steps in cyanogenic glycoside biosynthesis. The approach could guide functional characterization of other membrane-bound P450s and provide structural guidelines for elucidation of key structure-function relationships of other plant P450s.

Sequence modifications and heterologous expression of eukaryotic cytochromes P450 in Escherichia coli

The development of a heterologous expression system is often a principal step in biochemical and biotechnological studies on cytochromes P450 (P450s). However, heterologous expression of eukaryotic membrane-bound P450s in Escherichia coli is still a trial-and-error process because theoretical and systematical experimental procedures have not yet been established. In this study, we generated a series of chimeric variants of fungal P450s by replacing their N-terminal domains with the N-terminal domains of other P450s and explored their potentials for heterologous expression in E.coli. Large-scale screening demonstrated that a number of fungal P450s could be expressed when their N-terminal amino acid sequences were replaced with the corresponding domain of CYP5144C1, even when the expression of the non-chimeric sequence was unpromising. Furthermore, a comprehensive screening resulted in the identification of 64 different types of chimeric partners whose N-terminal domains could potentially be used to increase the expression levels of various P450s. These findings will help to elaborate experimental strategies for high-level heterologous expression of a variety of eukaryotic membrane-bound P450s in E.coli.

High-level heterologous expression of fungal cytochrome P450s in Escherichia coli

A thorough understanding of the sequence–structure–function relationships of cytochrome P450 (P450) is necessary to better understand the metabolic diversity of living organisms. Significant amounts of pure enzymes are sometimes required for biochemical studies, and their acquisition often relies on the possibility of their heterologous expression. In this study, we performed extensive heterologous expression of fungal P450s in Escherichia coli using 304 P450 isoforms. Using large-scale screening, we confirmed that at least 27 P450s could be expressed with/without simple sequence deletion at the 5′ end of cDNAs, which encode the N-terminal hydrophobic domain of the enzyme. Moreover, we identified N-terminal amino acid sequences that can potentially be used to construct chimeric P450s, which could dramatically improve their expression levels even when the expression of the wild-type sequence was unpromising. These findings will help increase the chance of heterologous expression of a variety of fungal and other eukaryotic membrane-bound P450s in E. coli.

High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy

Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzymes in humans, also catalyze important synthetic and detoxicative reactions in insects, plants and many microbes. Flexibilities in their catalytic sites and membrane associations are thought to play central roles in substrate binding and catalytic specificity. To date, Escherichia coli expression strategies for structural analysis of eukaryotic membrane-bound P450s by X-ray crystallography have necessitated full or partial removal of their N-terminal signal anchor domain and, often, replacement of residues more peripherally associated with the membrane (such as the F-G loop region). Even with these modifications, investigations of P450 structural flexibility remain challenging with multiple single crystal conditions needed to identify spatial variations between substrate-free and different substrate-bound forms. To overcome these limitations, we have developed methods for the efficient expression of 13C- and 15N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of co-expressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis 13C, 15N-labeled His 4CYP98A3 is expressed at yields of 2–4 mg per liter of minimal media without the necessity of generating side chain modifications or N-terminal deletions. Precipitated His 4CYP98A3 generates high quality SSNMR spectra consistent with a homogeneous, folded protein. These data highlight the potential of these methodologies to contribute to the structural analysis of membrane-bound proteins.

CYP153A6, a soluble P450 oxygenase catalyzing terminal-alkane hydroxylation.

The first and key step in alkane metabolism is the terminal hydroxylation of alkanes to 1-alkanols, a reaction catalyzed by a family of integral-membrane diiron enzymes related to Pseudomonas putida GPo1 AlkB, by a diverse group of methane, propane, and butane monooxygenases and by some membrane-bound cytochrome P450s. Recently, a family of cytoplasmic P450 enzymes was identified in prokaryotes that allow their host to grow on aliphatic alkanes. One member of this family, CYP153A6 from Mycobacterium sp. HXN-1500, hydroxylates medium-chain-length alkanes (C6 to C11) to 1-alkanols with a maximal turnover number of 70 min(-1) and has a regiospecificity of > or =95% for the terminal carbon atom position. Spectroscopic binding studies showed that C6-to-C11 aliphatic alkanes bind in the active site with Kd values varying from approximately 20 nM to 3.7 microM. Longer alkanes bind more strongly than shorter alkanes, while the introduction of sterically hindering groups reduces the affinity. This suggests that the substrate-binding pocket is shaped such that linear alkanes are preferred. Electron paramagnetic resonance spectroscopy in the presence of the substrate showed the formation of an enzyme-substrate complex, which confirmed the binding of substrates observed in optical titrations. To rationalize the experimental observations on a molecular scale, homology modeling of CYP153A6 and docking of substrates were used to provide the first insight into structural features required for terminal alkane hydroxylation.

An NMR‐Based Binding Assay Used To Screen For Compounds That Bind To Cytochrome P450 Enzymes

The cytochromes P450 are the enzymes predominantly responsible for the metabolism of drugs and other exogenous compounds that enter into our bodies. Because of these enzymes’ prominent role, it would be helpful to be able to quickly predict which compounds—potential drug candidates, toxins, or protoxins—bind to these enzymes, thus providing invaluable data to serve as the starting point for later, more thorough studies. This quick prediction can be accomplished in the form of a high-throughput assay that involves monitoring the 13C-attached 1H chemical shift (13C-attached 1H δ) and 13C δ of 13CH3NC (methyl isocyanide). As is the case for any short-chain alkylisocyanide, methyl isocyanide binds through its isonitrile moiety to the heme iron contained within the active site of the cytochrome P450 enzymes. We show here, in a series of 13C-1H HSQC spectra and for several cytochrome P450s, that the two previously mentioned chemical shifts of this ligand change in going from the free (unbound) form to the bound form and then, again upon addition of a known substrate (distinct from both bound and unbound). In addition, for the two full-length, membrane-bound P450s, we show that the chemical shifts indicative of the bound form change depending on whether the protein has been solubilized in 20% (v/v) glycerol or incorporated into DPC micelles.

Engineering Microsomal Cytochrome P450 2C5 to Be a Soluble, Monomeric Enzyme: MUTATIONS THAT ALTER AGGREGATION, PHOSPHOLIPID DEPENDENCE OF CATALYSIS, AND MEMBRANE BINDING

Deletion of the N-terminal membrane-spanning domain from microsomal P450s 2C5 and 2C3 generates the enzymes, 2C5dH and 2C3dH, that exhibit a salt-dependent association with membranes indicating that they retain a monofacial membrane interaction domain. The two proteins are tetramers and dimers, respectively, in high salt buffers, and only 2C5dH requires phospholipids to reconstitute fully the catalytic activity of the enzyme. Amino acid residues derived from P450 2C3dH between residues 201 and 210 were substituted for the corresponding residues in P450 2C5 to identify those that would diminish the membrane interaction, the phospholipid dependence of catalysis, and aggregation of 2C5dH. Each of four substitutions, N202H, I207L, S209G, and S210T, diminished the aggregation of P450 2C5dH and produced a monomeric enzyme. The N202H and I207L mutations also diminished the stimulation of catalytic activity by phospholipid and reduced the binding of P450 2C5dH to phospholipid vesicles. The modified enzymes exhibit rates of progesterone 21-hydroxylation that are similar to that of P450 2C5dH. These conditionally membrane-bound P450s with improved solubility in high salt buffers are suitable for crystallization and structural determination by x-ray diffraction studies.

39] Approaches to crystallizing P450s

Publisher Summary This chapter summarizes the methods that have been used to crystallize P450s and considers the strategies for crystallizing the membrane bound P450s. One approach for crystallizing eukaryotic P450s is to use site-directed mutagenesis for the purpose of producing soluble, monodisperse P450s. The inclusion of glycerol or other polyols also should be included in crystallization trials. The commercial crystallization kits are highly recommended. Another new and important tool that shows some promise is light scattering. New commercial devices are available that enable a rapid screen of protein solutions to check the state of aggregation and whether or not the sample is monodisperse. As membrane-bound P450s may have a tendency to aggregate, a check on the state of aggregation prior to setting up a large number of crystallization trials can save a considerable amount of time and frustration.

A three-dimensional protein model for human cytochrome P450 2D6 based on the crystal structures of P450 101, P450 102, and P450 108.

Cytochromes P450 (P450s) constitute a superfamily of phase I enzymes capable of oxidizing and reducing various substrates. P450 2D6 is a polymorphic enzyme, which is absent in 5-9% of the Caucasian population as a result of a recessive inheritance of gene mutations. This deficiency leads to impaired metabolism of a variety of drugs. All drugs metabolized by P450 2D6 contain a basic nitrogen atom, and a flat hydrophobic region coplanar to the oxidation site which is either 5 or 7 A away from the basic nitrogen atom. The aim of this study was to build a three-dimensional structure for the protein and more specifically for the active site of P450 2D6 in order to determine the amino acid residues possibly responsible for binding and/ or catalytic activity. Furthermore, the structural features of the active site can be implemented into the existing small molecule substrate model, thus enhancing its predictive value with respect to possible metabolism by P450 2D6. As no crystal structures are yet available for membrane-bound P450s (such as P450 2D6), the crystal structures of bacterial (soluble) P450 101 (P450cam), P450 102 (P450BM3), and P450 108 (P450terp) have been used to build a three-dimensional model for P450 2D6 with molecular modeling techniques. Several important P450 2D6 substrates were consecutively docked into the active site of the protein model. The energy optimized positions of the substrates in the protein agreed well with the original relative positions of the substrates within the substrate model. This confirms the usefulness of small molecule models in the absence of structural protein data. Furthermore, the derived protein model indicates new leads for experimental validation and extension of the substrate model.

Molecular Engineering of Microsomal P450 2a-4 to a Stable, Water-Soluble Enzyme

Peptitergented P450 2a-4 (Pepti-P450), a water-soluble form of the mouse microsomal P450 2a-4, was genetically engineered and expressed in Escherichia coli. The NH 2-terminal hydrophobic sequence (positions 2 to 19) of Pepti-P450 was replaced by a peptitergent PD 1, amphipathic peptide consisting of 24 residues (C. E. Schafmeister, L. J. Miercke, and R. M. Stroud (1993) Science 262, 734-738). The expression level of Pepti-P450 (90,000 molecules/cell) was at least four times greater than that of wild-type P450 2a-4. Since Pepti-P450 was quite stable and was expressed as a peripheral membrane protein, it can be easily purified from the membrane fraction treated with Na 2CO 3 without using any detergents during the chromatographic steps. The purified Pepti-P450 retained the spectral and catalytic properties of the unmodified enzyme with a similar K m value for steroid 15α-hydroxylase activity (19.7 μM in comparison to 14.2 μM of the wildtype). Gel permeation chromatography showed that the purified Pepti-P450 in the detergent-free buffer was an oligomer with an approximate molecular mass of 450 kDa. The replacement of the hydrophobic anchor domain with an amphipathic helix such as peptitergent, therefore, may provide a general method for engineering membrane-bound P450s to soluble enzymes.

A preliminary 3D model for cytochrome P450 2D6 constructed by homology model building

A homology model building study of cytochrome P450 2D6 has been carried out based on the crystal structure of cytochrome P450 101. The primary sequences of P450 101 and P450 2D6 were aligned by making use of an automated alignment procedure. This alignment was adjusted manually by matching alpha-helices (C, D, G, I, J, K and L) and beta-sheets (beta 3/beta 4) of P450 101 that are proposed to be conserved in membrane-bound P450s (Ouzounis and Melvin [Eur. J. Biochem., 198 (1991) 307]) to the corresponding regions in the primary amino acid sequence of P450 2D6. Furthermore, alpha-helices B, B' and F were found to be conserved in P450 2D6. No significant homology between the remaining regions of P450 101 and P450 2D6 could be found and these regions were therefore deleted. A 3D model of P450 2D6 was constructed by copying the coordinates of the residues from the crystal structure of P450 101 to the corresponding residues in P450 2D6. The regions without a significant homology with P450 101 were not incorporated into the model. After energy-minimization of the resulting 3D model of P450 2D6, possible active site residues were identified by fitting the substrates debrisoquine and dextrometorphan into the proposed active site. Both substrates could be positioned into a planar pocket near the heme region formed by residues Val370, Pro371, Leu372, Trp316, and part of the oxygen binding site of P450 2D6. Furthermore, the carboxylate group of either Asp100 or Asp301 was identified as a possible candidate for the proposed interaction with basic nitrogen atom(s) of the substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Hybrid cytochromes P-450 identify a substrate binding domain in P-450IIC5 and P-450IIC4.

The cytochrome P-450 superfamily of enzymes catalyzes the oxidative metabolism of innumerable lipophilic compounds (e.g., drugs, carcinogens, steroids). Although the three-dimensional structure of a soluble bacterial P-450 (P-450cam) has been solved, little is known about the structures of the membrane-bound mammalian P-450s. Thus, the structural features of these enzymes that determine their multisubstrate specificity are unknown. In this report, we identify a segment of the primary structure of the structurally similar but functionally distinct cytochromes P-450IIC5 and P-450IIC4, which determines the apparent affinity of these cytochromes for the conversion of progesterone into the mineralocorticoid deoxycorticosterone. P-450IIC5 exhibits a greater than 10-fold lower apparent Km than P-450IIC4 for progesterone 21-hydroxylation. Chimeric cDNAs were constructed and expressed in COS-1 cells, which encode hybrids between these enzymes. The hybrid enzymes were assayed for catalytic activity and compared to the parental proteins. A segment of P-450IIC5 was identified that conferred the lower Km of P-450IIC5 to P-450IIC4. Sequential reduction of the length of the exchanged segments led to a hybrid enzyme with a high affinity derived largely from P-450IIC4, which contains three amino acid residues derived from P-450IIC5 clustered between positions 113 and 118. This suggests that this region is part of a substrate binding domain. This region maps by alignment of amino acid sequences to a residue of P-450cam, which has been implicated in substrate binding, suggesting that these segments of the primary structure serve a similar functional role in these two distantly related proteins.

Expression of rat liver microsomal cytochrome P-450 in yeast.

Recent advanced technologies of molecular cloning and protein chemistry revealed the entire primary structure of various cytochrome P-450 species so that the multiplicity of P-450 was evidently clarified. The domains responsible for heme binding and subcellular localization were posturated on the basis of the comparison of their primary structures.The rat microsomal P-450MC cDNA was efficiently expressed in the yeast Sacchromyces cerevisiae under the control of alcohol dehydrogenase I promoter and terminator. The P-450MC protein synthesized in yeast was localized in microsomes, contained heme in the molecule and interacted with an yeast electron-transport chain to exhibit monooxygenase activity.The yeast host-vector system combined with the site-directed mutagenesis and chimeric gene construction can be useful for overproduction and characterization of membrane-bound P-450s.