Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: insights into substrate specificity and membrane bound structure-function

Cytochrome P450C24A1 (CYP24A1), a peripheral inner mitochondrial membrane hemoprotein and candidate oncogene, regulates the side-chain metabolism and biological function of vitamin D and many of its related analog drugs. Rational mutational analysis of rat CYP24A1 based on hybrid (2C5/BM-3) homology modeling and affinity labeling studies clarified the role of key domains (N-terminus, A', A, and F-helices, beta3a strand, and beta5 hairpin) in substrate binding and catalysis. The scope of our study was limited by an inability to purify stable mutant enzyme targeting soluble domains (B', G, and I-helices) and suggested greater conformational flexibility among CYP24A1's membrane-associated domains. The most notable mutants developed by modeling were V391T and I500A, which displayed defective-binding function and profound metabolic defects for 25-hydroxylated vitamin D3 substrates similar to a non-functional F-helix mutant (F249T) that we previously reported. Val-391 (beta3a strand) and Ile-500 (beta5 hairpin) are modeled to interact with Phe-249 (F-helix) in a hydrophobic cluster that directs substrate-binding events through interactions with the vitamin D cis-triene moiety. Prior affinity labeling studies identified an amino-terminal residue (Ser-57) as a putative active-site residue that interacts with the 3beta-OH group of the vitamin D A-ring. Studies with 3-epi and 3-deoxy-1,25(OH)2D3 analogs confirmed interactions between the 3beta-OH group and Ser-57 effect substrate recognition and trafficking while establishing that the trans conformation of A-ring hydroxyl groups (1alpha and 3beta) is obligate for high-affinity binding to rat CYP24A1. Our work suggests that CYP24A1's amphipathic nature allows for monotopic membrane insertion, whereby a pw2d-like substrate access channel is formed to shuttle secosteroid substrate from the membrane to the active-site. We hypothesize that CYP24A1 has evolved a unique amino-terminal membrane-binding motif that contributes to substrate specificity and docking through coordinated interactions with the vitamin D A-ring.     

Structure-function analysis of human cytochrome P450 3A4 using 7-alkoxycoumarins as active-site probes

The oxidation of a series of seven alkyl ethers of 7-hydroxycoumarin by cytochrome P450 3A4 (CYP3A4) has been studied to probe the active site of the enzyme. TLC of the reaction mixture showed formation of metabolites other than 7-hydroxycoumarin. The separation and characterization of the different metabolites of the C4 to C7 compounds were achieved using a combination of TLC, HPLC, and gas chromatography-electron impact mass spectra. Among the 7-alkoxycoumarins, 7-hexoxycoumarin was found to be the most suitable candidate for investigating the active site of cytochrome CYP3A4, due to the well-separated metabolite peaks on TLC and HPLC. 7-hexoxycoumarin was found to produce three side-chain hydroxylated products besides 7-hydroxycoumarin: 7-(5-hydroxyhexoxy)coumarin, 7-(4-hydroxyhexoxy)coumarin, and 7-(3-hydroxycoumarin). The substitution of residues from substrate recognition sites -1, -4, -5, and -6 of CYP3A4 showed a strong influence on the product profile of 7-hexoxycoumarin, the most prominent effects observed with mutants at residues 119, 301, 305, 370, 373, and 479. The docking of 7-hexoxycoumarin into a molecular model of CYP3A4 also confirmed the presence of these residues within 5 A of the substrate. A comparative study of cytochrome P450 2B1 showed that the active-site mutants F206L, T302V, V363A, and S478G but not V363L exhibited a dramatic decrease in total 7-hexoxycoumarin hydroxylation. The study suggests that although the electronic nature of the substrate is important, enzymatic constraints significantly contribute to CYP3A4 selectivity.     

Mutations of Trp275 and Trp397 altered the binding selectivity of Vibrio carchariae chitinase A

Point mutations of the active-site residues Trp168, Tyr171, Trp275, Trp397, Trp570 and Asp392 were introduced to Vibrio carchariae chitinase A. The modeled 3D structure of the enzyme illustrated that these residues fully occupied the substrate binding cleft and it was found that their mutation greatly reduced the hydrolyzing activity against pNP-[GlcNAc]₂ and colloidal chitin. Mutant W397F was the only exception, as it instead enhanced the hydrolysis of the pNP substrate to 142% and gave no activity loss towards colloidal chitin. The kinetic study with the pNP substrate demonstrated that the mutations caused impaired K_m and k_cat values of the enzyme. A chitin binding assay showed that mutations of the aromatic residues did not change the binding equilibrium. Product analysis by thin layer chromatography showed higher efficiency of W275G and W397F in G4–G6 hydrolysis over the wild type enzyme. Though the time course of colloidal chitin hydrolysis displayed no difference in the cleavage behavior of the chitinase variants, the time course of G6 hydrolysis exhibited distinct hydrolytic patterns between wild-type and mutants W275G and W397F. Wild type initially hydrolyzed G6 to G4 and G2, and finally G2 was formed as the major end product. W275G primarily created G2–G5 intermediates, and later G2 and G3 were formed as stable products. In contrast, W397F initially produced G1–G5, and then the high-M_r intermediates (G3–G5) were broken down to G1 and G2 end products. This modification of the cleavage patterns of chitooligomers suggested that residues Trp275 and Trp397 are involved in defining the binding selectivity of the enzyme to soluble substrates.     

Enzymatic determinants of the substrate specificity of CYP2C9: role of B'-C loop residues in providing the pi-stacking anchor site for warfarin binding

Previous modeling efforts have suggested that coumarin ligand binding to CYP2C9 is dictated by electrostatic and pi-stacking interactions with complementary amino acids of the protein. In this study, analysis of a combined CoMFA-homology model for the enzyme identified F110 and F114 as potential hydrophobic, aromatic active-site residues which could pi-stack with the nonmetabolized C-9 phenyl ring of the warfarin enantiomers. To test this hypothesis, we introduced mutations at key residues located in the putative loop region between the B' and C helices of CYP2C9. The F110L, F110Y, V113L, and F114L mutants, but not the F114Y mutant, expressed readily, and the purified proteins were each active in the metabolism of lauric acid. The V113L mutant metabolized neither (R)- nor (S)-warfarin, and the F114L mutant alone displayed altered metabolite profiles for the warfarin enantiomers. Therefore, the effect of the F110L and F114L mutants on the interaction of CYP2C9 with several of its substrates as well as the potent inhibitor sulfaphenazole was chosen for examination in further detail. For each substrate examined, the F110L mutant exhibited modest changes in its kinetic parameters and product profiles. However, the F114L mutant altered the metabolite ratios for the warfarin enantiomers such that significant metabolism occurred for the first time on the putative C-9 phenyl anchor, at the 4'-position of (R)- and (S)-warfarin. In addition, the Vmax for (S)-warfarin 7-hydroxylation decreased 4-fold and the Km was increased 13-fold by the F114L mutation, whereas kinetic parameters for lauric acid metabolism, a substrate which cannot interact with the enzyme by a pi-stacking mechanism, were not markedly affected by this mutation. Finally, the F114L mutant effected a greater than 100-fold increase in the Ki for inhibition of CYP2C9 activity by sulfaphenazole. These data support a role for B'-C helix loop residues F114 and V113 in the hydrophobic binding of warfarin to CYP2C9, and are consistent with pi-stacking to F114 for certain aromatic ligands.     

Functional differences in the cytochrome P450 1 family enzymes from Zebrafish (Danio rerio) using heterologously expressed proteins

Mammalian cytochrome P450 1 (CYP1) genes are well characterized, but in other vertebrates only the functions of CYP1A genes have been well studied. We determined the catalytic activity of zebrafish CYP1A, CYP1B1, CYP1C1, CYP1C2, and CYP1D1 proteins using 11 fluorometric substrates and benzo[a]pyrene (BaP). The resorufin-based substrates, 7-ethoxyresorufin, 7-methoxyresorufin, and 7-benzyloxyresorufin, were well metabolized by all CYP1s except CYP1D1. CYP1A metabolized nearly all substrates tested, although rates for non-resorufin substrates were typically lower than resorufin-based substrates. Zebrafish CYP1s did not metabolize 7-benzyloxyquinoline, 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin or 7-methoxy-4-(aminomethyl)-coumarin. CYP1B1 and CYP1C2 had the highest rates of BaP metabolism. 3-Hydroxy-BaP was a prominent metabolite for all but CYP1D1. CYP1A showed broad specificity and had the highest metabolic rates for nearly all substrates. CYP1C1 and CYP1C2 had similar substrate specificity. CYP1D1 had very low activities for all substrates except BaP, and a different regioselectivity for BaP, suggesting that CYP1D1 function may be different from other CYP1s.     

cDNA cloning and characterization of feline CYP1A1 and CYP1A2

Deficiency of drug glucuronidation in the cat is one of the major reasons why this animal is highly sensitive to the side effects of drugs. The characterization of cytochrome P450 isoforms belonging to the CYP1A subfamily, which exhibit important drug oxidation activities such as activation of pro-carcinogens, was investigated. Two cDNAs, designated CYP1A-a and CYP1A-b, corresponding to the CYP1A subfamily were obtained from feline liver. CYP1A-a and CYP1A-b cDNAs comprise coding regions of 1554 bp and 1539 bp, and encode predicted amino acid sequences of 517 and 512 residues, respectively. These amino acid sequences contain a heme-binding cysteine and a conserved threonine. The cDNA identities, as well as the predicted amino acid sequences containing six substrate recognition sites, suggest that CYP1A-a and CYP1A-b correspond to CYP1A1 and CYP1A2, respectively. This was confirmed by the kinetic parameters of the arylhydrocarbon hydroxylase and 7-ethoxyresorufin O-deethylase activities of expressed CYPs in yeast AH22 cells and by the tissue distribution of each mRNA. However, theophylline 3-demethylation is believed to be catalyzed by CYP1A1 in cats, based on the high V(max) and low K(m) seen, in contrast to other animals. Because feline CYP1A2 had a higher K(m) for phenacetin O-deethylase activity with acetaminophen, which cannot be conjugated with glucuronic acid due to UDP-glucuronosyltransferase deficiency, it is supposed that the side effects of phenacetin as a result of toxic intermediates are severe and prolonged in cats.     

Mutations of Trp275 and Trp397 altered the binding selectivity of Vibrio carchariae chitinase A

Point mutations of the active-site residues Trp168, Tyr171, Trp275, Trp397, Trp570 and Asp392 were introduced to Vibrio carchariae chitinase A. The modeled 3D structure of the enzyme illustrated that these residues fully occupied the substrate binding cleft and it was found that their mutation greatly reduced the hydrolyzing activity against pNP-[GlcNAc](2) and colloidal chitin. Mutant W397F was the only exception, as it instead enhanced the hydrolysis of the pNP substrate to 142% and gave no activity loss towards colloidal chitin. The kinetic study with the pNP substrate demonstrated that the mutations caused impaired K(m) and k(cat) values of the enzyme. A chitin binding assay showed that mutations of the aromatic residues did not change the binding equilibrium. Product analysis by thin layer chromatography showed higher efficiency of W275G and W397F in G4-G6 hydrolysis over the wild type enzyme. Though the time course of colloidal chitin hydrolysis displayed no difference in the cleavage behavior of the chitinase variants, the time course of G6 hydrolysis exhibited distinct hydrolytic patterns between wild-type and mutants W275G and W397F. Wild type initially hydrolyzed G6 to G4 and G2, and finally G2 was formed as the major end product. W275G primarily created G2-G5 intermediates, and later G2 and G3 were formed as stable products. In contrast, W397F initially produced G1-G5, and then the high-M(r) intermediates (G3-G5) were broken down to G1 and G2 end products. This modification of the cleavage patterns of chitooligomers suggested that residues Trp275 and Trp397 are involved in defining the binding selectivity of the enzyme to soluble substrates.     

Analysis of human cytochrome P450 2C8 substrate specificity using a substrate pharmacophore and site-directed mutants

The structural determinants of substrate specificity of human liver cytochrome P450 2C8 (CYP2C8) were investigated using site-directed mutants chosen on the basis of a preliminary substrate pharmacophore and a three-dimensional (3D) model. Analysis of the structural features common to CYP2C8 substrates exhibiting a micromolar K(m) led to a substrate pharmacophore in which the site of oxidation by CYP2C8 is 12.9, 8.6, 4.4, and 3.9 A from features that could establish ionic or hydrogen bonds, and hydrophobic interactions with protein amino acid residues. Comparison of this pharmacophore with a 3D model of CYP2C8 constructed using the X-ray structure of CYP2C5 suggested potential CYP2C8 amino acid residues that could be involved in substrate recognition. Twenty CYP2C8 site-directed mutants were constructed and expressed in yeast to compare their catalytic activities using five CYP2C8 substrates that exhibit different structures and sizes [paclitaxel, fluvastatin, retinoic acid, a sulfaphenazole derivative (DMZ), and diclofenac]. Mutation of arginine 241 had marked effects on the hydroxylation of anionic substrates of CYP2C8 such as retinoic acid and fluvastatin. Serine 100 appears to be involved in hydrogen bonding interactions with a polar site of the CYP2C8 substrate pharmacophore, as shown by the 3-4-fold increase in the K(m) of paclitaxel and DMZ hydroxylation after the S100A mutation. Residues 114, 201, and 205 are predicted to be in close contact with substrates, and their mutations lead either to favorable hydrophobic interactions or to steric clashes with substrates. For instance, the S114F mutant was unable to catalyze the 6alpha-hydroxylation of paclitaxel. The S114F and F205A mutants were the best catalysts for retinoic acid and paclitaxel (or fluvastatin) hydroxylation, respectively, with k(cat)/K(m) values 5 and 2.1 (or 2.4) times higher, respectively, than those found for CYP2C8. Preliminary experiments of docking of the substrate into the experimentally determined X-ray structure of substrate-free CYP2C8, which became available quite recently [Schoch, G. A., et al. (2004) J. Biol. Chem. 279, 9497], were consistent with key roles for S100, S114, and F205 residues in substrate binding. The results suggest that the effects of mutation of arginine 241 on anionic substrate hydroxylation could be indirect and result from alterations of the packing of helix G with helix B'.     

Dissecting the Microscopic Steps of the Cyclophilin A Enzymatic Cycle on the Biological HIV-1 Capsid Substrate by NMR

Peptidyl-prolyl isomerases (PPIases) are emerging as key regulators of many diverse biological processes. Elucidating the role of PPIase activity in vivo has been challenging because mutagenesis of active-site residues not only reduces the catalytic activity of these enzymes but also dramatically affects substrate binding. Employing the cyclophilin A PPIase together with its biologically relevant and natively folded substrate, the N-terminal domain of the human immunodeficiency virus type 1 capsid (CA^N) protein, we demonstrate here how to dissect residue-specific contributions to PPIase catalysis versus substrate binding utilizing NMR spectroscopy. Surprisingly, a number of cyclophilin A active-site mutants previously assumed to be strongly diminished in activity toward biological substrates based only on a peptide assay catalyze the human immunodeficiency virus capsid with wild-type activity but with a change in the rate-limiting step of the enzymatic cycle. The results illustrate that a quantitative analysis of catalysis using the biological substrates is critical when interpreting the effects of PPIase mutations in biological assays.     

Structure-based design of a highly active vitamin D hydroxylase from Streptomyces griseolus CYP105A1

CYP105A1 from Streptomyces griseolus has the capability of converting vitamin D 3 (VD 3) to its active form, 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) by a two-step hydroxylation reaction. Our previous structural study has suggested that Arg73 and Arg84 are key residues for the activities of CYP105A1. In this study, we prepared a series of single and double mutants by site-directed mutagenesis focusing on these two residues of CYP105A1 to obtain the hyperactive vitamin D 3 hydroxylase. R84F mutation altered the substrate specificity that gives preference to the 1alpha-hydroxylation of 25-hydroxyvitamin D 3 over the 25-hydroxylation of 1alpha-hydroxyvitamin D 3, opposite to the wild type and other mutants. The double mutant R73V/R84A exhibited 435- and 110-fold higher k cat/ K m values for the 25-hydroxylation of 1alpha-hydroxyvitamin D 3 and 1alpha-hydroxylation of 25-hydroxyvitamin D 3, respectively, compared with the wild-type enzyme. These values notably exceed those of CYP27A1, which is the physiologically essential VD 3 hydroxylase. Thus, we successfully generated useful enzymes of altered substrate preference and hyperactivity. Structural and kinetic analyses of single and double mutants suggest that the amino acid residues at positions 73 and 84 affect the location and conformation of the bound compound in the reaction site and those in the transient binding site, respectively.     

Thermodynamic fidelity of the mammalian cytochrome P450 2B4 active site in binding substrates and inhibitors

Structural plasticity of mammalian cytochromes P450 (CYP) has recently been explored in our laboratory and elsewhere to understand the ligand-binding promiscuity. CYP2B4 exhibits very different conformations and thermodynamic signatures in binding the small inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) versus the large bifonazole. Using four key active-site mutants (F296A, T302A, I363A, and V367L) that are involved in binding one or both inhibitors, we dissected the thermodynamic basis for the ability of CYP2B4 to bind substrates and inhibitors of different sizes and chemistry. In all cases, 1:1 binding stoichiometry was observed. The inhibitors 4-CPI, 1-(4-chlorophenyl)imidazole, and 1-(2-(benzyloxy)ethyl)imidazole bind to the mutants with a free energy difference (ΔΔG) of ∼ 0.5 to 1 kcal/mol compared with the wild type but with a large entropy-enthalpy compensation of up to 50 kcal/mol. The substrate testosterone binds to all four mutants with a ΔΔG of ∼ 0.5 kcal/mol but with as much as 40 kcal/mol of entropy-enthalpy compensation. In contrast, benzphetamine binding to V367L and F296A is accompanied by a ΔΔG of ∼ 1.5 and 3 kcal/mol, respectively. F296A, I363A, and V367L exhibit very different benzphetamine metabolite profiles, indicating the different substrate-binding orientations in the active site of each mutant. Overall, the findings indicate that malleability of the active site allows mammalian P450s to exhibit a high degree of thermodynamic fidelity in ligand binding.     

A rational approach to Re-engineer cytochrome P450 2B1 regioselectivity based on the crystal structure of cytochrome P450 2C5

The regioselectivity for progesterone hydroxylation by cytochrome P450 2B1 was re-engineered based on the x-ray crystal structure of cytochrome P450 2C5. 2B1 is a high K(m) progesterone 16alpha-hydroxylase, whereas 2C5 is a low K(m) progesterone 21-hydroxylase. Initially, nine individual 2B1 active-site residues were changed to the corresponding 2C5 residues, and the mutants were purified from an Escherichia coli expression system and assayed for progesterone hydroxylation. At 150 microm progesterone, I114A, F297G, and V363L showed 5-15% of the 21-hydroxylase activity of 2C5, whereas F206V showed high activity for an unknown product and a 13-fold decrease in K(m). Therefore, a quadruple mutant, I114A/F206V/F297G/V363L (Q), was constructed that showed 60% of 2C5 progesterone 21-hydroxylase activity and 57% regioselectivity. Based on their 2C5-like testosterone hydroxylation profiles, S294D and I477F alone and in combination were added to the quadruple mutant. All three mutants showed enhanced regioselectivity (70%) for progesterone 21-hydroxylation, whereas only Q/I477F had a higher k(cat). Finally, the remaining three single mutants, V103I, V367L, and G478V, were added to Q/I477F and Q/S294D/I477F, yielding seven additional multiple mutants. Among these, Q/V103I/S294D/I477F showed the highest k(cat) (3-fold higher than that of 2C5) and 80% regioselectivity for progesterone 21-hydroxylation. Docking of progesterone into a three-dimensional model of this mutant indicated that 21-hydroxylation is favored. In conclusion, a systematic approach to convert P450 regioselectivity across subfamilies suggests that active-site residues are mainly responsible for regioselectivity differences between 2B1 and 2C5 and validates the reliability of 2B1 models based on the crystal structure of 2C5.     

Identification of Val117 and Arg372 as critical amino acid residues for the activity difference between human CYP2A6 and CYP2A13 in coumarin 7-hydroxylation

Human cytochrome P450 (CYP) 2A6 and 2A13 play an important role in catalyzing the metabolism of many environmental chemicals including coumarin, nicotine, and several tobacco-specific carcinogens. Both CYP2A6 and CYP2A13 proteins are composed of 494 amino acid residues. Although CYP2A13 shares a 93.5% identity with CYP2A6 in the amino acid sequence, it is only about one-tenth as active as CYP2A6 in catalyzing coumarin 7-hydroxylation. To identify the key amino acid residues that account for such a remarkable difference, we generated a series of CYP2A6 and CYP2A13 mutants by site-directed mutagenesis/heterologous expression and compared their coumarin 7-hydroxylation activities. In CYP2A6, the amino acid residues at position 117 and 372 are valine (Val) and arginine (Arg), respectively; whereas in CYP2A13, they are alanine (Ala) and histidine (His). Kinetic analysis revealed that the catalytic efficiency (Vmax/Km) of the CYP2A6 Val(117)--> Ala and Arg(372)--> His mutants was drastically reduced (0.41 and 0.64 versus 3.23 for the wild-type CYP2A6 protein). In contrast, the catalytic efficiency of the CYP2A13 Ala(117) --> Val and His(372) --> Arg mutants was greatly increased (2.65 and 2.60 versus 0.31 for wild-type CYP2A13 protein). These results clearly demonstrate that the Val at position 117 and Arg at position 372 are critical amino acid residues for coumarin 7-hydroxylation. Based on the crystal structure of CYP2C5, we have generated the homology models of CYP2A6 and CYP2A13 and docked the substrate coumarin to the active site. Together with the kinetic characterization, our structural modeling provides explanations for the amino acid substitution results and the insights of detailed enzyme-substrate interactions.     

Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

Caffeic acid is a biologically active molecule that has various beneficial properties, including antioxidant, anticancer, and anti-inflammatory activities. In this study, we explored the catalytic potential of a bacterial cytochrome P450, CYP199A2, for the biotechnological production of caffeic acid. When the CYP199A2 enzyme was reacted with p-coumaric acid, it stoichiometrically produced caffeic acid. The crystal structure of CYP199A2 shows that Phe at position 185 is situated directly above, and only 6.35 Å from, the heme iron. This F185 residue was replaced with hydrophobic or hydroxylated amino acids using site-directed mutagenesis to create mutants with novel and improved catalytic properties. In whole-cell assays with the known substrate of CYP199A2, 2-naphthoic acid, only the wild-type enzyme hydroxylated 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibited a preference for C-5 hydroxylation. Interestingly, several F185 mutants (F185V, F185L, F185I, F185G, and F185A mutants) also acquired the ability to hydroxylate cinnamic acid, which was not hydroxylated by the wild-type enzyme. These results demonstrate that F185 is an important residue that controls the regioselectivity and the substrate specificity of CYP199A2. Furthermore, Escherichia coli cells expressing the F185L mutant exhibited 5.5 times higher hydroxylation activity for p-coumaric acid than those expressing the wild-type enzyme. By using the F185L whole-cell catalyst, the production of caffeic acid reached 15 mM (2.8 g/liter), which is the highest level so far attained in biotechnological production of this compound.     

NDM-1 Zn1-binding residue His116 plays critical roles in antibiotic hydrolysis

Bacteria expressing NDM-1 have been labeled as superbugs because it confers upon them resistance to a broad range of β-lactam antibiotics. The enzyme has a di?zinc active centre, with the Zn2 site extensively studied. The roles of active-site Zn1 ligand residues are, however, still not fully understood. We carried out structure-function studies using the mutants, H116A, H116N, and H116Q. Zinc content analysis showed that Zn1 binding was weakened by 40 to 60% in the H116 mutants. The enzymatic-activity studies showed that the lower hydrolysis rates were mainly caused by their weaker substrate binding. The catalytic efficiency (k_cat/K_m) of the mutants followed the order: WT > > H116Q (decreased by 4–20 fold) > H116A (decreased by 20–700 fold) ≥ H116N (decreased by 6–800 fold). The maximum effect was observed on H116N against penicillin G, whereas ampicillin was not hydrolyzed at all. The fold-increase of K_m values, which informs the weakening of substrate binding, were: H116A by 5–45 fold; H116N by 6–100 fold; H116Q by 2–10 fold. Molecular dynamics simulations suggested that the Zn1 site mutations affected the positions of Zn2 and the bridging hydroxide, by 0.8 to 1.2 Å, with the largest changes of ~1.5 Å observed on Zn2 ligand C221. A native hydrogen bond between H118 and D236 was disrupted in the H116N and H116Q mutants, which led to increased flexibility of loop 10. Consequently, residue N233 was no longer maintained at an optimal position for substrate binding. H116 connected loop 7 across Zn1 to loop 10, thereby contributed to the overall integrity. This work revealed that the H116-Zn1 interaction plays a critical role in defining the substrate-binding site. From these results, it can be inferred that inhibition strategies targeting the zinc ions may be a new direction for drug development.     

Crystal Structure of CYP24A1, a Mitochondrial Cytochrome P450 Involved in Vitamin D Metabolism

Cytochrome P450 (CYP) 24A1 catalyzes the side-chain oxidation of the hormonal form of vitamin D. Expression of CYP24A1 is up-regulated to attenuate vitamin D signaling associated with calcium homeostasis and cellular growth processes. The development of therapeutics for disorders linked to vitamin D insufficiency would be greatly facilitated by structural knowledge of CYP24A1. Here, we report the crystal structure of rat CYP24A1 at 2.5 Å resolution. The structure exhibits an open cleft leading to the active-site heme prosthetic group on the distal surface that is likely to define the path of substrate access into the active site. The entrance to the cleft is flanked by conserved hydrophobic residues on helices A′ and G′, suggesting a mode of insertion into the inner mitochondrial membrane. A docking model for 1α,25-dihydroxyvitamin D₃ binding in the open form of CYP24A1 that clarifies the structural determinants of secosteroid recognition and validates the predictive power of existing homology models of CYP24A1 is proposed. Analysis of CYP24A1's proximal surface identifies the determinants of adrenodoxin recognition as a constellation of conserved residues from helices K, K″, and L that converge with an adjacent lysine-rich loop for binding the redox protein. Overall, the CYP24A1 structure provides the first template for understanding membrane insertion, substrate binding, and redox partner interaction in mitochondrial P450s.     

Structural comparison of cytochromes P450 2A6, 2A13, and 2E1 with pilocarpine

Human xenobiotic-metabolizing cytochrome P450 (CYP) enzymes can each bind and monooxygenate a diverse set of substrates, including drugs, often producing a variety of metabolites. Additionally, a single ligand can interact with multiple CYP enzymes, but often the protein structural similarities and differences that mediate such overlapping selectivity are not well understood. Even though the CYP superfamily has a highly canonical global protein fold, there are large variations in the active site size, topology, and conformational flexibility. We have determined how a related set of three human CYP enzymes bind and interact with a common inhibitor, the muscarinic receptor agonist drug pilocarpine. Pilocarpine binds and inhibits the hepatic CYP2A6 and respiratory CYP2A13 enzymes much more efficiently than the hepatic CYP2E1 enzyme. To elucidate key residues involved in pilocarpine binding, crystal structures of CYP2A6 (2.4 ?), CYP2A13 (3.0 ?), CYP2E1 (2.35 ?), and the CYP2A6 mutant enzyme, CYP2A6?I208S/I300F/G301A/S369G (2.1 ?) have been determined with pilocarpine in the active site. In all four structures, pilocarpine coordinates to the heme iron, but comparisons reveal how individual residues lining the active sites of these three distinct human enzymes interact differently with the inhibitor pilocarpine.     

Substrate recognition by family 7 alginate lyase from Sphingomonas sp. A1

Sphingomonas sp. A1 alginate lyase A1-II′, a member of polysaccharide lyase family 7, shows a broad substrate specificity acting on poly α-L-guluronate (poly(G)), poly β-D-mannuronate (poly(M)) and the heteropolymer (poly(MG)) in alginate molecules. A1-II′ with a glove-like β-sandwich as a basic scaffold forms a cleft covered with two lid loops (L1 and L2). Here, we demonstrate the loop flexibility for substrate binding and structural determinants for broad substrate recognition and catalytic reaction. The two loops associate mutually over the cleft through the formation of a hydrogen bond between their edges (Asn141 and Asn199). A double mutant, A1-II′ N141C/N199C, has a disulfide bond between Cys141 and Cys199, and shows little enzyme activity. Adding dithiothreitol to the enzyme reaction mixture leads to a tenfold increase in its molecular activity, suggesting the significance of flexibility in lid loops for accommodating the substrate into the active cleft. In alginate trisaccharide (GGG or MMG)-bound A1-II′ Y284F, the enzyme interacts appropriately with substrate hydroxyl groups at subsites + 1 and + 2 and accommodates G or M, while substrate carboxyl groups are strictly recognized by specific residues. This mechanism for substrate recognition enables A1-II′ to show the broad substrate specificity. The structure of A1-II′ H191N/Y284F complexed with a tetrasaccharide bound at subsites − 1 to + 3 suggests that Gln189 functions as a neutralizer for the substrate carboxyl group, His191 as a general base, and Tyr284 as a general acid. This is, to our knowledge, the first report on the structure and function relationship in family 7.     

Identification of human CYP2C19 residues that confer S-mephenytoin 4'-hydroxylation activity to CYP2C9

CYP2C19 is selective for the 4'-hydroxylation of S-mephenytoin while the highly similar CYP2C9 has little activity toward this substrate. To identify critical amino acids determining the specificity of human CYP2C19 for S-mephenytoin 4'-hydroxylation, we constructed chimeras by replacing portions of CYP2C9 containing various proposed substrate recognition sites (SRSs) with those of CYP2C19 and mutating individual residues by site-directed mutagenesis. Only a chimera containing regions encompassing SRSs 1--4 was active (30% of wild-type CYP2C19), indicating that multiple regions are necessary to confer specificity for S-mephenytoin. Mutagenesis studies identified six residues in three topological components of the proteins required to convert CYP2C9 to an S-mephenytoin 4'-hydroxylase (6% of the activity of wild-type CYP2C19). Of these, only the I99H difference located in SRS 1 between helices B and C reflects a change in a side chain that is predicted to be in the substrate-binding cavity formed above the heme prosthetic group. Two additional substitutions, S220P and P221T residing between helices F and G but not in close proximity to the substrate binding site together with five differences in the N-terminal portion of helix I conferred S-mephenytoin 4'-hydroxylation activity with a K(M) similar to that of CYP2C19 but a 3-fold lower K(cat). Three residues in helix I, S286N, V292A, and F295L, were essential for S-mephenytoin 4'-hydroxylation activity. On the basis of the structure of the closely related enzyme CYP2C5, these residues are unlikely to directly contact the substrate during catalysis but are positioned to influence the packing of substrate binding site residues and likely substrate access channels in the enzyme.     

Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR

The membrane heme protein cytochrome b₅ (b₅) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b₅ and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b₅, and an androgen-forming lyase reaction that is facilitated 10-fold by b₅. NMR chemical shift mapping of b₅ titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b₅ residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b₅ binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b₅ on the two CYP17A1-mediated reactions and, thus, communication between the superficial b₅ binding site and the buried CYP17A1 active site, CYP17A1/b₅ complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b₅ interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b₅ binding site.