Membrane binding and substrate access merge in cytochrome P450 7A1, a key enzyme in degradation of cholesterol.

To study membrane topology and mechanism for substrate specificity, we truncated residues 2-24 in microsomal cytochrome P450 7A1 (P450 7A1) and introduced conservative and nonconservative substitutions at positions 214-227. Heterologous expression in Escherichia coli was followed by investigation of the subcellular distribution of the mutant P450s and determination of the kinetic and substrate binding parameters for cholesterol. The results indicate that a hydrophobic region, comprising residues 214-227, forms a secondary site of attachment to the membrane in P450 7A1 in addition to the NH(2)-terminal signal-anchor sequence. There are two groups of residues at this enzyme-membrane interface. The first are those whose mutation results in more cytosolic P450 (Val-214, His-225, and Met-226). The second group are those whose mutation leads to more membrane-bound P450 (Phe-215, Leu-218, Ile-224, and Phe-227). In addition, the V214A, V214L, V214T, F215A, F215L, F215Y, L218I, L218V, V219T, and M226A mutants showed a 5-12-fold increased K(m) for cholesterol. The k(cat) of the V214A, V214L, V219T, and M226A mutants was increased up to 1.8-fold, and that of the V214T, F215A, F215L, F215Y, L218I, and L218V mutants was decreased 3-10.5-fold. Based on analysis of these mutations we suggest that cholesterol enters P450 7A1 through the membrane, and Val-214, Phe-215, and Leu-218 are the residues located near the point of cholesterol entry. The results provide an understanding of both the P450 7A1-membrane interactions and the mechanism for substrate specificity.

Putative helix F contributes to regioselectivity of hydroxylation in mitochondrial cytochrome P450 27A1.

On the basis of alignment with structurally characterized cytochromes P450 (P450s), we have identified the putative F and G helices of mitochondrial P450s 27A1 and 11A. We introduced substitutions at Phe-207, Ile-211, and Phe-215 within putative helix F and at Trp-235 and Tyr-238 within putative helix G in P450 27A1 and compared wild type and mutants with respect to catalytic activity, product pattern, substrate binding, formation of hydrogen peroxide, and interaction with redox partner. Results indicate that the mutated residues are important for delivery of the correctly oriented substrate to the P450 active site. The I211K and F215K mutations, for example, affected the regioselectivity of P450 27A1-dependent hydroxylation reactions and conferred the P450 capacity to cleave the C-C bond of the substrate during the catalytic cycle. Studies of P450 11A1 indicate that Phe-202 has functions similar to those of its counterpart in P450 27A1 (Phe-215). We propose that putative helices F and G form the sides of the substrate-access channel, thus providing the additional mechanism to control regioselectivity of hydroxylation in mitochondrial P450s.

Oxysterols in the circulation of patients with the Smith-Lemli-Opitz syndrome: abnormal levels of 24S- and 27-hydroxycholesterol.

Infants with the cholesterol synthesis defect Smith- Lemli-Opitz syndrome (SLO) have reduced activity of the enzyme 7-dehydrocholesterol-7-reductase and accumulate 7-dehydrocholesterol, with the highest concentration in the brain. As a result of the generally reduced content of cholesterol, plasma levels of oxysterols would be expected to be reduced. 24S-hydroxycholesterol is almost exclusively formed in the brain, whereas 27-hydroxycholesterol is mainly formed from extrahepatic and extracerebral cholesterol. In accordance with the expectations, sterol-correlated plasma levels of 24S-hydroxycholesterol were reduced in infants with SLO (by about 50%). In contrast, the sterol-correlated levels of 27-hydroxycholesterol in the circulation were markedly increased. No side-chain oxidized metabolites of 7-dehydrocholesterol were detected in the circulation. Recombinant human CYP27 had markedly lower 27-hydroxylase activity toward 7-dehydrocholesterol than towards cholesterol. HEK293 cells expressing 24S-hydroxylase active toward cholesterol had no significant activity towards 7-dehydrocholesterol. The plasma levels of 3 beta,7 alpha-dihydroxy-5-cholestenoic in the patients acid were reduced, suggesting a generally reduced metabolism of 27-oxygenated steroids. It is concluded that the accumulation of 7-dehydrocholesterol in the brains of patients with SLO reduces formation of 24S-hydroxycholesterol. The condition is associated with markedly increased circulating levels of 27-hydroxycholesterol, most probably due to reduced metabolism of this oxysterol. We discuss the possibility that the circulating levels of 24S-hydroxycholesterol may be used as a marker for the severity of the disease.--Björkhem, I., L. Starck, U. Andersson, D. Lütjohann, S. von Bahr, I. Pikuleva, A. Babiker, and U. Diczfaulsy. Oxysterols in the circulation of patients with the Smith-Lemli-Opitz syndrome: abnormal levels of 24S- and 27-hydroxycholesterol. J. Lipid Res. 2001. 42: 366--371.

The tertiary structure of full-length bovine adrenodoxin suggests functional dimers.

The three-dimensional X-ray crystal structure of full-length oxidized bovine adrenodoxin (Adx) has been determined at 2.5 A resolution by molecular replacement using a structure of a truncated form as a starting model. Crystals of Adx belong to a primitive monoclinic space group P2(1) with four Adx molecules in an asymmetric unit. The unit cell dimensions are a = 59.44 A, b = 77.03 A, c = 59.68 A, and beta = 94.83 degrees. The structure has been refined to an R factor of 23.5%. Structures of the four molecules of full-length Adx (127 amino acids) in the asymmetric unit were compared with each other and also with that of the truncated Adx (4-108). The overall topology of full-length Adx remains the same as described earlier for the truncated protein. Differences that do occur are almost wholly confined to alternate side-chain conformations that reflect differing lattice contacts made by two proteins. Extensive interactions found between molecules 1 and 2 in the full-length Adx asymmetric unit may reflect the ability of Adx to form dimers in vivo and are consistent with hydrodynamic measurements which show that in solution there is an equilibrium between monomeric and dimeric forms of Adx. Dimerization of Adx could explain why the truncated form has greater affinity for the P450 redox partner than the full-length form. From these results it can be considered that the mechanism of electron transfer is not necessarily the same in different mitochondrial P450 systems.

An additional electrostatic interaction between adrenodoxin and P450c27 (CYP27A1) results in tighter binding than between adrenodoxin and p450scc (CYP11A1).

Mitochondrial cytochrome P450c27 (product of the CYP27A1 gene) is found to have significantly higher affinity for the common redox partner adrenodoxin than another mitochondrial P450, P450scc (product of the CYP11A1 gene). To investigate the basis of the approximately 30-fold difference in adrenodoxin binding, two sets of P450c27 mutants were generated, expressed in Escherichia coli, and purified. Mutations of one set were within the putative adrenodoxin-binding site containing conserved lysine residues also crucial in P450scc for binding adrenodoxin. The second set included mutations within a sequence aligning with the "meander region" of P450BM-3 proposed to be a site of redox-partner interactions in P450s (Hasemann, C. A., Kurumbail, R. G., Boddupalli, S. S., Peterson, J. A., and Deisenhofer, J. (1995) Structure 3, 41-62). Mutation of the P450c27 conserved lysines (K354A and K358A) led to a approximately 20-fold increase in apparent Ks for adrenodoxin, confirming that these two positively charged residues conserved in mitochondrial P450s are important for adrenodoxin binding. Mutation of Arg-418, conserved in the CYP27A1 family, to serine also decreased the affinity for adrenodoxin approximately 20-fold. This residue is predicted to be located in the meander region. A triple K354A/K358A/R418S mutation profoundly reduced adrenodoxin binding. Thus, in contrast to P450scc, where mutation of the two conserved positively charged residues results in virtually complete inhibition of adrenodoxin binding, in P450c27 there are three of such residues (Lys-354, Lys-358, and Arg-418) important for adrenodoxin interaction.

Activities of recombinant human cytochrome P450c27 (CYP27) which produce intermediates of alternative bile acid biosynthetic pathways.

The primary physiological significance of cytochrome P450c27 (CYP27) has been associated with its role in the degradation of the side chain of C27 steroids in the hepatic bile acid biosynthesis pathway, which begins with 7alpha-hydroxylation of cholesterol in liver. However, recognition that in humans P450c27 is a widely or ubiquitously expressed mitochondrial P450, and that there are alternative pathways of bile acid synthesis which begin with 27-hydroxylation of cholesterol catalyzed by P450c27, suggests the need to reevaluate the role of this enzyme and its catalytic properties. 27-Hydroxycholesterol was thought to be the only product formed upon reaction of P450c27 with cholesterol. However, the present study demonstrates that recombinant human P450c27 is also able to further oxidize 27-hydroxycholesterol giving first an aldehyde and then 3beta-hydroxy-5-cholestenoic acid. Kinetic data indicate that in a reconstituted system, after 27-hydroxycholesterol is formed from cholesterol, it is released from the P450 and then competes with cholesterol for reentry the enzyme active site for further oxidation. Under subsaturating substrate concentrations, the efficiencies of oxidation of 27-hydroxycholesterol and 3beta-hydroxy-5-cholestenal to the acid by human P450c27 are greater than the efficiency of hydroxylation of cholesterol to 27-hydroxycholesterol indicating that the first hydroxylation step in the overall conversion of cholesterol into 3beta-hydroxy-5-cholestenoic acid is rate-limiting. Interestingly, 3beta-hydroxy-5-cholestenoic acid was found to be further metabolized by the recombinant human P450c27, giving two monohydroxylated products with the hydroxyl group introduced at different positions on the steroid nucleus.

Escherichia coli flavodoxin sepharose as an affinity resin for cytochromes P450 and use to identify a putative cytochrome P450c17/3beta-hydroxysteroid dehydrogenase interaction.

Flavodoxin Sepharose (Fld Sepharose), a reagent originally developed to demonstrate an interaction between native Escherichia coli Fld and cytochrome P450c17, has been synthesized, using highly expressed (7 micromol Fld/liter E. coli culture) recombinant E. coli Fld, for use as an affinity resin for microsomal cytochromes P450. As a test of the specificity of Fld Sepharose, we have examined the utility of this resin for purification of P450c17 and P450c21 from a relatively crude mixture of solubilized adrenocortical microsomal proteins. Chromatography of this mixture on Fld Sepharose resulted in a threefold enrichment of cytochrome P450 specific content without spectrally detectable P450 denaturation. Electrophoretic and immunoblot analyses of fractions eluted from the Fld Sepharose column revealed the presence of P450c17 and P450c21, both of which were sufficiently pure, after SDS-PAGE, for identification by N-terminal sequence analysis. Intriguingly, a major protein copurifying with P450c17 and P450c21 was identified as 3beta-hydroxysteroid dehydrogenase (3beta-HSD) which was subsequently found not to directly bind Fld Sepharose. Purified bovine 3beta-HSD covalently linked to Sepharose can bind recombinant bovine P450c17, an interaction which is partially disrupted upon mild heat denaturation of P450c17 or by the nonionic detergent Emulgen. This interaction, however, does not appear to affect P450c17 hydroxylase and lyase activities as measured in vitro. From these results, we propose that 3beta-HSD and P450c17 may associate, perhaps as part of a steroidogenic complex, in the endoplasmic reticulum.

Expression, purification, and enzymatic properties of recombinant human cytochrome P450c27 (CYP27).

A large number of microsomal P450s have been expressed in Escherichia coli in quantities sufficient for structure/function analysis. However, only one mitochondrial P450 has been successfully overexpressed, that being cholesterol side chain cleavage cytochrome P450 (P450scc). We report here overexpression, purification, and characterization of a second mitochondrial P450, human sterol C-27 hydroxylase (P450c27). The conditions used for expression are very similar to those applied for P450scc, although a quite different purification protocol was necessary to achieve highly purified P450c27. The catalytic properties of purified recombinant human P450c27 resemble those of purified, endogenous rat and rabbit P450c27, regarding both specificity and turnover numbers. Like endogenous P450c27 from rat and rabbit liver, human recombinant P450c27 is only functional in the presence of adrenodoxin and adrenodoxin reductase and shows no activity in the presence of the microsomal P450 reductase. We conclude that P450c27 is most likely not the 1alpha-hydroxylase of 25-hydroxyvitamin D3, contrary to a previous suggestion (Axen, E., Postlind, H., Sjöberg, H., and Wikvall, K. (1994) Proc. Natl. Acad. Sci. USA 91, 10014-10018) because this activity of P450c27 (28 pmol/min/nmol P450) seems far too low to be physiologically relevant. This activity is 10(3) times lower than the 27-hydroxylase activity toward 5beta-cholestane-3alpha,7alpha,12alpha-triol and 40 times lower than the 27-hydroxylation of cholesterol by this enzyme. The development of this expression system and purification procedure creates the potential for structure/function analysis of P450c27, including possible crystallization of this important enzyme.

Studies of distant members of the P450 superfamily (P450scc and P450c27) by random chimeragenesis.

"Random chimeragenesis" (Kim, J.-Y., and Devreotes, P. N. (1994) J. Biol. Chem. 269, 28724-28731; Levin, L. R., and Reed, R. R. (1995) J. Biol. Chem. 270, 7573-7579) has been used to generate chimeras between two distant P450s involved in catabolism of cholesterol in mammals, P450scc (product of CYP11A1 gene) and P450c27 (product of CYP27 gene). Both are mitochondrial P450s which hydroxylate the side chain of cholesterol. Even though these P450s are only about 25% identical, we wondered whether their similar substrate specificity might permit mapping of the active sites by this technique. Four chimeric DNAs encoding three different proteins have been obtained testifying that short stretches of nucleotide identity (six nucleotides preceding the crossover point) are sufficient for homologous recombination in Escherichia coli, the basis of the random chimeragenesis technique. The N-terminal part of the chimeras was formed by P450scc and the C-terminus was from P450c27. The chimeric P450s have been expressed in E. coli and partially purified. Though they displayed a peak at 453 nm in their CO-difference spectra, indicating that the proteins are properly folded and hemin is correctly incorporated, none of the chimeras had detectable catalytic activity with either cholesterol (substrate for P450scc) or 5beta-cholestane-3alpha,7alpha,12alpha-triol (substrate for P450c27). To investigate whether the chimeras bind substrate, titrations with 22R-hydroxycholesterol have been carried out. Addition of 22R-hydroxycholesterol to recombinant P450scc which is in the low spin form leads to conversion to the high spin form. Titration of chimeras with 22R-hydroxycholesterol did not result in their conversion to the high spin form. However, a shift to 426-430 nm was observed in the difference spectrum of two of the chimeras with a minimum around 406 nm. Thus, addition of 22R-hydroxycholesterol to these chimeras results in binding of the steroid in the enzyme active site and conversion of one low spin form to a different low spin form. Possible explanations for the absence of enzymatic activity and the potential advantages of the random chimeragenesis technique to generate chimeras between different P450s are discussed.

Genetically engineered bacterial cells and applications.

Heterologous expression systems have proven to be very important in P450 research, permitting investigation of many P450s identified only by recombinant (cloning) technologies. They also make it possible to study human P450s since tissue sources for naturally occurring enzymes are difficult to obtain. A variety of different heterologous systems have been applied to the study of P450s and each one has its own unique advantages. For the study of biophysical properties and structure/function relationships, E. coli has proven to be a particularly good system. Both microsomal and mitochondrial P450s can be expressed to high levels in E. coli and subsequently purified to homogeneity for detailed analysis. Techniques of both site directed mutagenesis and random chimeragenesis are very facile in bacteria providing excellent opportunity to analyze specific aspects of structure/function relationships of P450s. Furthermore microsomal P450s are active in intact E. coli, activities being supported by flavodoxin and flavodoxin reductase, providing the opportunity to develop bioreactors expressing designer P450s. The salient features of P450 expression in bacteria are summarized herein.

Active-site topology of bovine cholesterol side-chain cleavage cytochrome P450 (P450scc) and evidence for interaction of tyrosine 94 with the side chain of cholesterol.

Combining site-directed mutagenesis with analysis of the active-site topology of bovine cholesterol side-chain cleavage cytochrome P450scc (P450scc), we have investigated the roles of tyrosine residues 93 and 94 on substrate binding. Four single mutants (Y93A, Y93S, Y94A, and Y94S) and one double mutant (Y93S/Y94S) were examined. The largest increase in Ks was observed for binding of cholesterol and 25-hydroxycholesterol to the Y94S mutant (approximately 5.5-fold), with a smaller increase (< 2.5-fold) for binding of 22-hydroxycholesterol. Mutation of Y94 thus appears to influence the interaction with cholesterol, 25-hydroxycholesterol, and possibly 22-hydroxycholesterol. Y93 is not involved in binding of 22- and 25-hydroxycholesterol but may interact with cholesterol. The active-site topologies of P450scc and its mutants were probed by reaction with three arylhydrazines. The N-arylprotoporphyrin IX regioisomer patterns obtained with phenyl- and 2-naphthylhydrazine indicate that the active site is primarily open above pyrrole ring A and suggest that a region some distance above pyrrole ring D is also open. The single mutations Y93S, Y93A, Y94A, and Y94S do not detectably alter the regioisomer patterns obtained with the phenyl- and 2-naphthyl probes, but a small, reproducible change is observed with the 2-naphthyl probe for the Y93S/Y94S double mutant. The conformational alteration implied by this change could not be detected by titration with 22- and 25-hydroxycholesterol but is detectable by titration with cholesterol. The results indicate that cholesterol binds over pyrrole ring D of the heme in bovine P450scc, strongly suggest that Y94 interacts with the side chain of cholesterol, and provide evidence that the side chains of 22- and 25-hydroxycholesterol bind to a different region of the active site than the side chain of cholesterol.

Functional reconstitution of cytochrome P-450scc with hemin activated with Woodward's reagent K. Formation of a hemeprotein cross-link.

In order to evaluate structure-function relationships of heme moiety in cytochrome P-450scc, we carried out the reconstitution of apoprotein with Fe-protoporphyrin IX, one carboxyl group of which was converted to reactive enol ester by Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate). Woodward's reagent K can be used as a cross-linking reagent, since amino groups can apparently react with the enol ester. Treatment of cytochrome P-450scc with H2O2 was used to obtain the apoprotein. Functional reconstitution of the hemin derivative with apocytochrome P-450scc was achieved. The reconstituted hemeprotein was purified, and the resulting preparation contained no P-420 form and had the same cholesterol-hydroxylating activity as a control preparation. 30% of the reconstituted hemin was covalently bound to protein. Heme-linked peptide (Gly177-Phe194) was isolated. Its possible role in the active site formation of cytochrome P-450scc is discussed.

Chemical modification of rabbit IgG with N-dansylaziridine. Investigation of the properties of dansylated antibodies.

To elucidate the effect of antigen binding fluorescent thiol reagent, N-dansylaziridine (DAZ), which is sensitive to the changes in the microenvironment, was used for modification of rabbit IgG hinge region cysteine residue. DAZ binds to hinge region Cys 226 as evidenced by the structural analysis. Labelling of IgG with DAZ does not alter its conformation, hydrodynamic behavior, nor its antigen binding properties. Upon antigen bindings, the fluorescence intensity of modified IgG increases about 80%. This result indicates that interaction of antibodies with antigen is accompanied by the conformational changes in the IgG hinge region.

[Chemical modification of rabbit immunoglobulin G by dansylaziridine and study of the properties of modified antibodies]. / Khimicheskaia modifikatsiia immunoglobulinov G krolika N-dansilaziridinom i issledovanie svoistv modifitsirovannykh antitel.

To elucidate the effect of the antigen binding fluorescent thiol reagent, N-dansylaziridine (DAZ) which is sensitive to microenvironmental changes, was used for modification of the rabbit IgG hinge region cystine residue. DAZ binds to the hinge region Cys 226 as could be evidenced from the structural analysis data. Labelling of IgG with DAZ does not alter either its conformation and hydrodynamic behaviour or its antigen binding properties. Upon antigen binding the fluorescence intensity of modified IgG increases up to about 80%. This finding suggests that the interaction of antibodies with the antigen is accompanied by conformational changes in the IgG hinge region.

[Chemical modification of cholesterol-hydroxylating cytochrome P-450 from adrenal cortex mitochondria with diethylpyrocarbonate]. / Khimicheskaia modifikatsiia kholesteringidroksiliruiushchego tsitokhroma P-450 mitokhondrii kory nadpochechnikov diétilpirokarbonatom.

Chemical modification of adrenocortical cytochrome P-450scc with diethyl pyrocarbonate has been carried out. The histidine residues and the protein amino groups were shown to undergo modification. Carbethoxylation was accompanied by the hemoprotein inactivation and the loss of enzymatic activity. Neither of the high spin effectors (i.e., substrate and adrenodoxin) protected cytochrome P-450scc either from inactivation or from the loss of enzymatic activity. The data obtained are discussed in terms of the functional role of histidine residues in the cytochrome P-450scc molecule.

[A new method of preparing hemin conjugates with rabbit IgG]. / Novyi sposob polucheniia kon''iugatov gemina s IgG krolika.

Stable derivatives of hemin activated by means of the Woodward reagent K were obtained, and the reaction conditions for their covalent attachment to the IgG molecule optimized. Advantages of the new method for conjugation are discussed.

Selective chemical modification of cytochrome P-450SCC lysine residues. Identification of lysines involved in the interaction with adrenodoxin.

Selective chemical modification of cytochrome P-450SCC has been carried out with lysine-modifying reagents. Modification of cytochrome P-450SCC with succinic anhydride was shown to result in loss of its ability to interact with intermediate electron transfer protein - adrenodoxin. To identify amino acid residues involved in charge-ion pairing with complementary carboxyl groups of adrenodoxin, cytochrome P-450SCC complex with adrenodoxin was modified with succinic anhydride. Adrenodoxin was then removed and cytochrome P-450 was additionally modified with isotopically labelled reagent. Subsequent chymotryptic hydrolysis of [14C]succinylated cytochrome P-450SCC and separation of digest obtained by combining various types of HPLC resulted in seven major radioactive peptides. The amino acid sequence of the peptides was determined by microsequencing. The major amino groups modified with radioactive succinic anhydride were found to be at Lys-73, -109, -110, -126, -145, -148 and -154 in the N-terminal sequence of cytochrome P-450SCC molecule and at Lys-267, -270, -338 and -342 in the C-terminal sequence. The role of electrostatic interactions in fixation of cytochrome P-450SCC complex with adrenodoxin is discussed.

[Study of the role of lysine residues of the cholesterol hydroxylating cytochrome P-450 by a method of chemical modification]. / Issledovanie roli ostatkov lizina kholesteringidroksiliruiushchego tsitrokhroma P-450 metodom khimicheskoi modifikatsii.

Chemical modification of cytochrome P-450scc by lysine-specific reagents has been performed. Modification of the hemoprotein was shown to result in the loss of its ability to interact with adrenodoxin. With a view of identifying lysine residues involved in the interaction with adrenodoxin, cytochrome P-450scc was modified by succinic anhydride in the presence of adrenodoxin. After the removal of ferredoxin, the modification was performed with the use of a radioactively labeled reagent. Subsequent hydrolysis of the succinic hemoprotein by chymotrypsin and separation of the peptides obtained by high pressure liquid chromatography resulted in the isolation of seven chymotryptic peptides containing labeled lysine residues. These amino acid sequences were identified. The role of lysine residues of cytochrome P-450scc in complex formation with adrenodoxin is discussed.