Structure-activity correlations in pentachlorobenzene oxidation by engineered cytochrome P450cam

We had reported engineering of the heme monooxygenase cytochrome P450cam from Pseudomonas putida with the F87W/Y96F/L244A/V247L mutations for the oxidation of pentachlorobenzene (PeCB), a recalcitrant environmental contaminant, to pentachlorophenol. In order to provide further insights into P450 structure, function and substrate recognition, we have determined the crystal structure of this 4-mutant without a substrate and its complex with PeCB. PeCB is bound face-on to the heme, with a weak Fe--Cl interaction. One PeCB chlorine is located in the cavity generated by the L244A mutation, in striking illustration of the role of this mutation in promoting PeCB binding. The structures also show that the P450(cam) oxygen-binding groove between G248 and T252 is flexible and can tolerate significant deviations from their conformations in the wild type without loss of enzyme activity. Analysis of the PeCB binding interactions led to introduction of the T101A mutation to enable the substrate to reorient during the catalytic cycle for more efficient oxidation. The resultant 5-mutant F87W/Y96F/T101A/L244A/V247L is 3-fold more active for PeCB oxidation than the 4-mutant. Polychlorinated benzene binding by the mutants and the partitioning between substrate oxidation and non-productive (uncoupling) side reactions are correlated with the structural data.     

Intramolecular electron transfer in a cytochrome P450cam system with a site-specific branched structure

Cytochrome P450 (P450) is an attractive oxygenase due to the diverse catalytic reactions and the broad substrate specificity. Class I P450s require an excess concentration (more than 10 times) of iron-sulfur proteins, which transfer electrons to P450s, to attain the maximum catalytic activity and this requirement is a critical bottleneck for practical applications. Here, we show a site-specific branched fusion protein of P450 with its electron transfer proteins using enzymatic cross-linking with transglutaminase. A branched fusion protein of P450 from Pseudomonas putida (P450cam), which was composed of one molecule each of P450cam, putidaredoxin (Pdx) and Pdx reductase, showed higher catalytic activity (306 min(-1)) and coupling efficiency (99%) than the equimolar reconstitution system due to the intramolecular electron transfer. The unique site-specific branched structure simply increased local concentration of proteins without denaturation of each protein. Therefore, enzymatic post-translational protein manipulation can be a powerful alternative to conventional strategies for the creation of multicomponent enzyme systems with novel proteinaceous architecture.     

A predicted three-dimensional structure of human cytochrome P450: implications for substrate specificity

A three-dimensional structure for human cytochrome P450IA1 waspredicted based on the crystal coordinates of cytochrome P450camfrom Pseudomonas putida. As there was only 15% residue identitybetween the two enzymes, additional information was used toestablish an accurate sequence alignment that is a prerequisitefor model building. Twelve representative eukaryotic sequenceswere aligned and a net prediction of secondary structure wasmatched against the known -helices and ß-sheets ofP450cam. The cam secondary structure provided a fixed main-chainframework onto which loops of appropriate length from the humanP450IA1 structure were added. The model-built structure of thehuman cytochrome conformed to the requirements for the segregationof polar and nonpolar residues between the core and the surface.The first 44 residues of human cytochrome P450 could not bebuilt into the model and sequence analysis suggested that residues1–26 formed a single membrane-spanning segment. Examinationof the sequences of cytochrome P450s from distinct gene familiessuggested specific residues that could account for the differencesin substrate specificity. A major substrate for P450IA1, 3-methyl-cholanthrene,was fitted into the proposed active site and this planar aromaticmolecule could be accommodated into the available cavity. Residuesthat are likely to interact with the haem were identified. Thesequence similarity between 59 eukaryotic enzymes was representedas a dendrogram that in general clustered according to genefamily. Until a crystallographic structure is available, thismodel-building study identifies potential residues in cytochromeP450s important in the function of these enzymes and these residuesare candidates for site-directed mutagenesis.     

Comparison of the construction of a 3-D model for human thromboxane synthase using P450cam and BM-3 as templates: implications for the substrate binding pocket

A 3-D model of human thromboxane A₂ synthase (TXAS) was constructedusing a homology modeling approach based on information fromthe 2.0 crystal structure of the hemoprotein domains of cytochromeP450BM-3 and P450cam. P450BM-3 is a bacterial fatty acid monooxygenaseresembling eukaryotic microsomal cytochrome P450s in primarystructure and function. TXAS shares 26.4% residue identity and48.4% residue similarity with the P450BM-3 hemoprotein domain.The homology score between TXAS and P450BM-3 is much higherthan that between TXAS and P450cam. Alignment between TXAS andthe P450BM-3 hemoprotein domain or P450cam was determined throughsequence searches. The P450BM-3 or P450cam main-chain coordinateswere spplied to the TXAS main chain in those sements where thetwo sequences were well aligned. These segments were linkedto one another using a fragment search method, and the sidechains were added to produce a 3-D model for TXAS. A TXAS substrate,prostaglandin H₂ (PGH₂) was docked into the TXAS cavity correspondingto the arachidonic acid binding pocket in P450BM-3 or camphorbinding site in P450cam. Regions of the heme and putative PGH2binding cavities in the TXAS model were identified and analyzed.The segments and residues involved in the active-site pocketof the TXAS model provide reasonable candidates for TXAS proteinengineering and inhibitor design. Comparison of the TXAS modelbased on P450BM-3 with another TXAS model based on the P450BM-3with another TXAS model based on the P450cam structure indicatedthat P450BM-3 is a more suitable template for homology modelingof TXAS.     

Construction and evaluation of a three-dimensional structure of cytochrome P450choP enzyme (CYP105C1)

The purpose of this work was to develop and carefully evaluateimproved strategies for constructing reliable 3-D models ofP450 isozymes. To this end, a unique combination of steps forbuilding and evaluating a model structure was used to builda homology model of the P450choP isozyme, based on knowledgeof the X-ray structures of P450cam, P450terp, P450BM-3 and P450eryF.Specifically, the reliability of this model was examined bysystematic comparisons of its conformational, energetic, environmentaland packing properties and those of the four reference proteinswith corresponding properties from the database of proteinswith known structures. The results showed that the examinedproperties of this model structure are well within the criteriaestablished for reliable structures and are of nearly as goodquality as those of the reference proteins. In addition, theresult from a 120 ps unconstrained MD simulation of the modelwith structural waters provided evidence that the model is stableat room temperature. This 3-D model can now be reliably usedfor explicit characterization of substrate and inhibitor complexes.Most importantly, although it is envisioned that building modelsfor mammalian P450s will be even more challenging, the stepsdescribed here should be very useful in future constructionof 3-D models of mammalian P450 isozymes.     

Ile115Leu mutation in the SRS1 region of an insect cytochrome P450 (CYP6B1) compromises substrate turnover via changes in a predicted product release channel

CYP6B1 represents the principal cytochrome P450 monooxygenase responsible for metabolizing furanocoumarins in Papilio polyxenes, an insect that specializes on host plants containing these toxins. Investigations of the amino acids responsible for the efficient metabolism of these plant toxins has identified Ile115 as one that modulates the rate of furanocoumarin metabolism even though it is predicted to be positioned at the edge of the heme plane and outside substrate contact regions. In contrast to previous expression studies conducted under conditions of limiting P450 reductase showing that the Ile115-to-Leu replacement enhances turnover of xanthotoxin and other furanocoumarins, studies conducted at high P450 reductase indicate that the Ile115-to-Leu replacement reduces turnover of these substrates. Further analysis of substrate binding affinities, heme spin state and NADPH consumption rates indicate that, whereas the I115L replacement mutant displays higher substrate affinity and heme spin state than the wild-type CYP6B1 protein, it utilizes NADPH more slowly than the wild-type CYP6B1 protein at high P450 reductase levels. Molecular models developed for the wild-type CYP6B1 and mutant protein suggest that more constricted channels extending from the catalytic site in the I115L mutant to the P450 surface limit the rate of product release from this mutant catalytic site under conditions not limited by the rate of electron transfer from NADPH.     

Substrate mobility in thiocamphor-bound cytochrome P450cam: an explanation of the conflict between the observed product profile and the X-ray structure

Thiocamphor is an unusual substrate for P450_cam in that in theX-ray structure it binds in the active site pocket in two distinctorientations and neither of these orientations are consistentwith the 5-alcohol being the primary product. Other camphoranalogs such as norcamphor or camphane bind in a single orientationconsistent with the 5-alcohol being a major product. We presentan analysis of four 175 ps molecular dynamics trajectories ofthiocamphor-bound cytochrome P450_cam. The first two trajectorieswere calculated for cytochrome P450_cam with thiocamphor boundin both its major and minor crystallographic orientations. Inthe second set of simulations, a single oxygen atom was addedas a distal ligand to the heme group in order to model the putativeferryl oxygen reaction intermediate. Trajectories were againcalculated starting with thiocamphor in its major and minororientations. While the protein dynamics were quite similarin all four trajectories, the substrate showed distinctly differentmotions in each of the trajectories. In particular, the preferredsubstrate orientations were very different in the presence ofthe ferryl oxygen than in the absence of that oxygen. The preferredorientations in the absence of the distal oxygen were consistentwith the 3-akohol being the major product, while the preferredorientations in the presence of the distal oxygen were consistentwith the 5-alcohol being a major product. These simulationsoffer an explanation for the inconsistency between the X-raydata and the product profile.     

The Metabolism of a Novel Cytochrome P450 (CYP77B34) in Tribenuron-Methyl-Resistant Descurainia sophia L. to Herbicides with Different Mode of Actions

Descurainia sophia L. (flixweeds) is a noxious broad-leaf weed infesting winter wheat fields in China that has evolved high resistance to tribenuron-methyl. In this work, a brand new gene CYP77B34 was cloned from tribenuron-methyl-resistant (TR) D. sophia and transferred into Arabidopsis thaliana, and the sensitivities of Arabidopsis with or without the CYP77B34 transgene to herbicides with a different mode of actions (MoAs) were tested. Compared to Arabidopsis expressing pCAMBIA1302-GFP (empty plasmid), Arabidopsis transferring pCAMBIA1302-CYP77B34 (recombinant plasmid) became resistant to acetolactate synthase (ALS)-inhibiting herbicide tribenuron-methyl, protoporphyrinogen oxidase (PPO)-inhibiting herbicides carfentrazone-ethyl and oxyfluorfen. Cytochrome P450 inhibitor malathion could reverse the resistance to tribenuron-methyl, carfentrazone-ethyl and oxyfluorfen in transgenic Arabidopsis plants. In addition, the metabolic rates of tribenuron-methyl in Arabidopsis expressing CYP77B34 were significantly higher than those in Arabidopsis expressing pCAMBIA1302-GFP. Other than that, the transgenic plants showed some tolerance to very-long-chain fatty acid synthesis (VLCFAs)-inhibiting herbicide pretilachlor and photosystem (PS) II-inhibiting herbicide bromoxynil. Subcellular localization revealed that the CYP77B34 protein was located in the endoplasmic reticulum (ER). These results clearly indicated that CYP77B34 mediated D. sophia resistance to tribenuron-methyl and may have been involved in D. sophia cross-resistance to carfentrazone-ethyl, oxyfluorfen, pretilachlor and bromoxynil.     

Generation of a Ni(II) binding site by introduction of a histidine cluster in the structure of human glutathione transferase Al-1

Two mutant forms of human glutathione transferase (GST) Al–1with affinity for metal ions were constructed by introductionof His residues by site–directed mutagenesis. A mutant,2–His, contained the mutations Lys84Gln, Asp85His andGlu88His, and another, 5–His, contained the mutationsTyr79His, Asn80His, Lys84His, Asp85His and Glu88HLs. The mutantproteins were obtained in good yields (40–150 mg per 3I culture) by heterologous expression in Escherichia coli. Themutant enzymes possessed novel binding affinities for Ni(II)and Zn(II) ions, as demonstrated by immobilized metal ion affinitychromatography. The mutant with two novel His residues (2–Hismutant) did not bind as tightly to immobilized Nifll) as didthe mutant with five novel His residues (5–His mutant).When tested for affinity to immobilized Zn(II), only the 5–Hismutant remained bound to the column. The affinity of the 5–Hismutant for Ni(II) ions in solution was determined by bindingexperiments in an aqueous polymeric two–phase system.Analysis of the binding curve showed two binding sites per enzymesubunit and a dissociation constant of 6.7 1 . 6 M. The kineticconstants k_cat, K_m and k_cat/k_m for the reaction with glutathioneand l–chloro–2,4–dinitrobenzene were determinedby steady–state kinetic analysis and the parameter valuesfor the mutant forms were found to be indistinguishable fromthose obtained for the wild–type GST Al–1. The differencesin surface charge in the mutant proteins as compared with thewild–type enzyme did not alter the pH dependence of k_cat.The results provide an alternative method for purification offully active recombinant GST Al–1 by the introductionof novel metal binding sites. The data also showed that twoHis residues are sufficient for Ni(II) binding.     

Loss of Protein Stability and Function Caused by P228L Variation in NADPH-Cytochrome P450 Reductase Linked to Lower Testosterone Levels

Cytochrome P450 oxidoreductase (POR) is the redox partner of steroid and drug-metabolising cytochromes P450 located in the endoplasmic reticulum. Mutations in POR cause a broad range of metabolic disorders. The POR variant rs17853284 (P228L), identified by genome sequencing, has been linked to lower testosterone levels and reduced P450 activities. We expressed the POR wild type and the P228L variant in bacteria, purified the proteins, and performed protein stability and catalytic functional studies. Variant P228L affected the stability of the protein as evidenced by lower unfolding temperatures and higher sensitivity to urea denaturation. A significant decline in the rate of electron transfer to cytochrome c and thiazolyl blue tetrazolium (MTT) was observed with POR P228L, while activities of CYP3A4 were reduced by 25% and activities of CYP3A5 and CYP2C9 were reduced by more than 40% compared with WT POR. The 17,20 lyase activity of CYP17A1, responsible for the production of the main androgen precursor dehydroepiandrosterone, was reduced to 27% of WT in the presence of the P228L variant of POR. Based on in silico and in vitro studies, we predict that the change of proline to leucine may change the rigidity of the protein, causing conformational changes in POR, leading to altered electron transfer to redox partners. A single amino acid change can affect protein stability and cause a severe reduction in POR activity. Molecular characterisation of individual POR mutations is crucial for a better understanding of the impact on different redox partners of POR.