Folding Requirements Are Different between Sterol 14α-Demethylase (CYP51) from Mycobacterium tuberculosis and Human or Fungal Orthologs
2001; Elsevier BV; Volume: 276; Issue: 30 Linguagem: Inglês
10.1074/jbc.m102767200
ISSN1083-351X
AutoresGalina I. Lepesheva, Larissa M. Podust, Aouatef Bellamine, Michael R. Waterman,
Tópico(s)Signaling Pathways in Disease
ResumoUpon sequence alignment of CYP51 sterol 14α-demethylase from animals, plants, fungi, and bacteria, arginine corresponding to Arg-448 of CYP51 in Mycobacterium tuberculosis (MT) is conserved near the C terminus of all family members. In MTCYP51 Arg-448 forms a salt bridge with Asp-287, connecting β-strand 3–2 with helix J. Deletion of the three C-terminal residues of MTCYP51 has little effect on expression of P450 in Escherichia coli. However, truncation of the fourth amino acid (Arg-448) completely abolishes P450 expression. We have investigated whether Arg-448 has other structural or functional roles in addition to folding and whether its conservation reflects conservation of a common folding pathway in the CYP51 family. Characterization of wild type protein and three mutants, R448K, R448I, and R448A, including examination of catalytic activity, secondary and tertiary structure analysis by circular dichroism and tryptophan fluorescence, and studies of both equilibrium and temporal MTCYP51 unfolding behavior, shows that Arg-448 does not play any role in P450 function or maintenance of the native structure. C-terminal truncation of Candida albicans and human CYP51 orthologs reveals that, despite conservation in sequence, the requirement for arginine at the homologous C-terminal position in folding in E. coli is not conserved. Thus, despite similar spatial folds, functionally related but evolutionarily distinct P450s can follow different folding pathways. Upon sequence alignment of CYP51 sterol 14α-demethylase from animals, plants, fungi, and bacteria, arginine corresponding to Arg-448 of CYP51 in Mycobacterium tuberculosis (MT) is conserved near the C terminus of all family members. In MTCYP51 Arg-448 forms a salt bridge with Asp-287, connecting β-strand 3–2 with helix J. Deletion of the three C-terminal residues of MTCYP51 has little effect on expression of P450 in Escherichia coli. However, truncation of the fourth amino acid (Arg-448) completely abolishes P450 expression. We have investigated whether Arg-448 has other structural or functional roles in addition to folding and whether its conservation reflects conservation of a common folding pathway in the CYP51 family. Characterization of wild type protein and three mutants, R448K, R448I, and R448A, including examination of catalytic activity, secondary and tertiary structure analysis by circular dichroism and tryptophan fluorescence, and studies of both equilibrium and temporal MTCYP51 unfolding behavior, shows that Arg-448 does not play any role in P450 function or maintenance of the native structure. C-terminal truncation of Candida albicans and human CYP51 orthologs reveals that, despite conservation in sequence, the requirement for arginine at the homologous C-terminal position in folding in E. coli is not conserved. Thus, despite similar spatial folds, functionally related but evolutionarily distinct P450s can follow different folding pathways. P450 gene or protein sterol 14α-demethylase cytochrome P450 sterol 14α-demethylase from M. tuberculosis high performance liquid chromatography dihydrolanosterol base pair(s) polymerase chain reaction 4-morpholinepropanesulfonic acid Cytochromes P450 (CYP)1form a large superfamily of monooxygenases found in organisms from protists to mammals (1Nelson D.R. Koymans L. Kamataki T. Stegeman J.J. Feyereisen R. Waxman D.J. Waterman M.R. Gotoh O. Coon M.J. Estabrook R.W. Gunsalus I.C. Nebert D.W. Pharmacogenetics. 1996; 1: 1-42Crossref Scopus (2662) Google Scholar). They catalyze oxidative synthesis and metabolism of various kinds of physiologically important lipophilic compounds, such as sterols, fatty acids, hormones, biosignaling substances or phytochemicals, as well as detoxify xenobiotics, such as drugs, food additives, and environmental contaminants (1Nelson D.R. Koymans L. Kamataki T. Stegeman J.J. Feyereisen R. Waxman D.J. Waterman M.R. Gotoh O. Coon M.J. Estabrook R.W. Gunsalus I.C. Nebert D.W. Pharmacogenetics. 1996; 1: 1-42Crossref Scopus (2662) Google Scholar, 2Belpaire F.M Bogaert M.G. Acta Clin. Belg. 1996; 51: 254-260Crossref PubMed Scopus (26) Google Scholar). It has been suggested that all P450s have evolved from a common, ancestral gene by duplication, followed by specific mutations that alter substrate specificity (3Nelson D.R. Kamataki T. Waxman D.J. Guengerich F.P. Estabrook R.W. Feyereisen R. Gonzalez F.J. Coon M.J. Gunsalus I.C. Gotoh O. Okuda K. Nebert D.W. DNA Cell Biol. 1993; 12: 1-51Crossref PubMed Scopus (1654) Google Scholar). Besides having vastly different substrates, cytochromes P450 differ in their intracellular localization and redox partners (4Omura T. Ishimura Y. Fujii-Kuriyama Y. Cytochrome P450. 2nd Ed. Kodansha, Tokyo1993Google Scholar). As a result, they often exhibit low (sometimes less than 20%) amino acid sequence identity (5Gotoh O. J. Biol. Chem. 1992; 267: 83-90Abstract Full Text PDF PubMed Google Scholar, 6Graham S.E. Peterson J.A. Arch. Biochem. Biophys. 1999; 369: 24-29Crossref PubMed Scopus (167) Google Scholar). However, comparative analysis of known cytochrome P450 crystal structures (6Graham S.E. Peterson J.A. Arch. Biochem. Biophys. 1999; 369: 24-29Crossref PubMed Scopus (167) Google Scholar, 7Hasemann C.A. Kurumbail R.G. Boddupalli S.S. Peterson J.A. Deisenhofer J. Structure. 1995; 3: 41-62Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar), hydropathy and secondary structure prediction algorithms (8Tretiakov V.E. Degtyarenko K.N. Uvarov V.Yu. Archakov A.I. Arch. Biochem. Biophys. 1989; 275: 429-439Crossref PubMed Scopus (24) Google Scholar), and molecular modeling (9Dai R. Pincus M.R. Friedman F.K. Cell. Mol. Life Sci. 2000; 57: 487-499Crossref PubMed Scopus (31) Google Scholar) indicates that all P450s have a very similar spatial fold. Among more than 1500 known forms of P450, sterol 14α-demethylase (CYP51) represents the only CYP gene occurring in different biological kingdoms with essentially the same metabolic role (10Aoyama Y. Noshiro M. Gotoh O. Imaoka S. Funae Y. Kurosawa N. Horiuchi T. Yoshida Y. J. Biochem. (Tokyo). 1996; 119: 926-933Crossref PubMed Scopus (108) Google Scholar). Via three successive monooxygenation reactions, it catalyzes the removal of the 14α-methyl group from cyclized precursors in sterol biosynthetic pathways (11Fisher R.T. Stam S.H. Johnson P.R. Ko S.S. Magolda R.L. Gaylor J.L. Trzaskos J.M. J. Lipid Res. 1989; 30: 1621-1632PubMed Google Scholar, 12Yoshida Y. Aoyama Y. Noshiro M. Gotoh O. Biochem. Biophys. Res. Commun. 2000; 273: 799-804Crossref PubMed Scopus (99) Google Scholar). In animals CYP51 participates in cholesterol biosynthesis. Fungal and plant isoforms are involved in the synthesis of ergosterol and phytosterols, respectively. Phylogenetic analyses based on protein sequence data have shown that eukaryotic CYP51s are joined into a distinctive evolutionary cluster with bacterial CYP51-like proteins (13Yoshida Y. Noshiro M. Aoyama Y. Kawamoto T. Horiuchi T. Gotoh O. J. Biochem. (Tokyo). 1997; 122: 1122-1128Crossref PubMed Scopus (76) Google Scholar). These findings led to a suggestion that CYP51 is one of the most ancient CYP families, which arose in the prokaryotic era before divergence of eukaryotic branches and has been distributed into major biological kingdoms concomitant with their diversification (12Yoshida Y. Aoyama Y. Noshiro M. Gotoh O. Biochem. Biophys. Res. Commun. 2000; 273: 799-804Crossref PubMed Scopus (99) Google Scholar). Identification of sterol 14α-demethylase in M. tuberculosis (14Aoyama Y. Horiuchi T. Gotoh O. Noshiro M. Yoshida Y. J. Biochem. (Tokyo). 1998; 124: 694-696Crossref PubMed Scopus (71) Google Scholar, 15Bellamine A. Mangla A.T. Nes W.D. Waterman M.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8937-8942Crossref PubMed Scopus (192) Google Scholar) has provided the opportunity for structural studies of one of the evolutionary oldest CYP gene products. Unlike eukaryotic isoforms, which are localized in the endoplasmic reticulum, MTCYP51 does not contain the N-terminal signal-anchor sequence and is water-soluble. This has made it possible to obtain protein crystals and to determine the MTCYP51 structure (16Podust L.M. Poulos T.L. Waterman M.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3068-3073Crossref PubMed Scopus (480) Google Scholar). In the MTCYP51 crystal structure, Arg-448 (β-strand 3–2) ties together different regions of the molecule by forming a salt bridge with Asp-287 (J-helix) and multiple weak interactions with main chain atoms of amino acid residues 412–414 (junction L-helix-β-strand 3–3). The role of Arg-448 in the MTCYP51 structure/function relationship has been investigated, and effects of C-terminal truncation in bacterial and eukaryotic (Candida albicans and human) isoforms of sterol 14α-demethylase have been compared. It is found that presence of Arg-448 or another amino acid at this position in MTCYP51 is essential for proper folding but not for maintenance of its structure or for catalytic function. However, in eukaryotic CYP51, the C terminus is not even required for folding. We conclude that, upon heterologous expression in bacterial cells, folding of prokaryotic and eukaryotic forms of CYP51 follows different pathways. Restriction endonucleases and other modifying enzymes were purchased from New England Biolabs (Beverly, MA). Reagents for bacterial growth were from Difco (Sparks, MD). 24-[3H]Dihydrolanosterol (DHL) was a generous gift from Dr. J. Trzaskos (DuPont Merck Pharmaceutical Co, Wilmington, DE), and Triton WR 1339 was from Serva (Heidelberg, Germany). Other chemicals were purchased from Sigma. Recombinant E. coli flavodoxin and flavodoxin reductase were expressed and purified as described previously (17Jenkins C.M. Waterman M.R. Biochemistry. 1998; 37: 6106-6113Crossref PubMed Scopus (79) Google Scholar). To insert a four-histidine tag at the N terminus of MTCYP51, the cDNA cloned in pET17b expression vector, Novagen (Madison, WI) (15Bellamine A. Mangla A.T. Nes W.D. Waterman M.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8937-8942Crossref PubMed Scopus (192) Google Scholar) was digested atNdeI-XbaI sites (−1 to 20 bp) and two annealed oligos, forward (5′-TATGCATCACCATCACAGCGCTGTTGCACTACC-3′) and reverse (5′-CCGGGGTAGTGCAACAGCGCTGTGATGGTGATGCA-3′), were ligated between the restriction sites (histidine codons are underlined). The ligation mixture was transformed into XL1 Blue cells (Stratagene, La Jolla, CA) and resultant colonies analyzed by PCR using forward T7 promoter primer, and reverse 5′-CCGGGGTAGTGCAACAGCGCTGTGATGGTGATGCA-3′ primer, which is complementary to N-His-tagged MTCYP51 sequence from 2 to 36 bp. Selected colonies were sequenced, and plasmid DNA containing four histidine codons following the initiator methionine was used as a template for site-directed mutagenesis of MTCYP51. As a template for mutagenesis of human CYP51, we used its previously described pCW expression vector (18Stromstedt M. Rozman D. Waterman M.R. Arch. Biochem. Biophys. 1996; 329: 73-81Crossref PubMed Scopus (154) Google Scholar). C. albicans CYP51 cDNA in YEp51 yeast expression vector was a generous gift from Dr. S. Kelly (University of Wales, Aberystwyth, United Kingdom) (19Shyadehi A.Z. Lamb D.C. Kelly S.L Kelly D.E. Schunck W.H. Wright J.N. Corina D. Akhtar M. J. Biol. Chem. 1996; 271: 12445-12450Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). To clone C. albicans CYP51 into pCW, 2M. Stromstedt, unpublished data. the cDNA was removed from the above yeast expression vector bySalI/HindIII restriction and inserted into the same sites in pBSIIKS (Stratagene). Two internal NdeI sites in the coding sequence (at 188 and 1117 bp) were destroyed by silent mutations. Then C. albicans CYP51 cDNA was amplified by PCR. The upstream primer 5′- GGAATTCCATATGGCTCTGTTATTAGCAGTTTTTGATGGCATT-3′ corresponding to the C. albicans CYP51 sequence from 1 to 33 bp, contained an NdeI site (underlined) and modification of N-terminal codons in order to optimize expression in E. colias done for human CYP51. The downstream primer 5′-CGCAAGCTT CAGTGATGGTGATGAAACATACAAGTTTCTCTTTTTTCCC-3′ from 23 to 49 bp is complementary to C. albicans CYP51 cDNA sequence from 1559 to 1584 bp. A HindIII site (underlined) followed by a stop codon (bold character) and four His codons were introduced. The resulting 1692-bp PCR product was isolated from agarose gel, digested by NdeI/HindIII, and cloned into pCW. The sequence in the region synthesized by PCR was confirmed by DNA sequencing. Site-directed mutagenesis was carried out using QuikChange™ site-directed mutagenesis kit (Stratagene) according to the supplier's instructions. The forward and reverse primers used to introduce mutations by PCR are shown in Table I. Mutations were confirmed by DNA sequencing.Table IOligonucleotides and templates used to generate M. tuberculosis, human, and C. albicans CYP51 mutantsTemplateMutationOligonucleotides1-aThe mutated nucleotides are underlined.pET17b/N-His MTCYP51T449Stop5′-GTGCGCTACCGCCGGCGATGAGGAGTTTGAAGCTTG-3′5′-GTACCAAGCTTCAAACTCCTCATCGCCGGCGGTAGCGCAC-3′R448Stop1-bCoding triplets for the mutants R448K, R448I, R448A, and R448D were AAA, ATC,GCT, and GAC, respectively, in place ofTGA.5′-CTTGCGTGCGCTACCGCCGGTGAACGGGAGTTTGAAGCTTG-3′5′-CAAGCTTCAAACTCCCGTTCACCGGCGGTAGCGCACGCAAG-3′D287R5′-GCCGTGATCGACGAACTCCGCGAGCTGTACGGCGACGGC-3′5′-GCCGTCGCCGTACAGCTCGCGGAGTTCGTCGATCACGGC-3′pCW/Hum CYP51R501Stop5′-CCAGTTATCCGTTACAAACGATGATCAAAACATCACCATCAC-3′5′-GTGATGGTGATGTTTTGATCATCGTTTGTAACGGATAACTGG-3′Y484Stop5′-CCCACTGTGAATTGAACAACTATGATTCACACCCC-3′5′-GGGGTGTGAATCATAGTTGTTCAATTCACAGTGGG-3′pCW/C.albCYP51R523Stop5′-GCAGAAATCATTTGGGAAAAATGAGAAACTTGTATGTTTCATC-3′5′-GATGAAACATACAAGTTTCTCATTTTTCCCAAATGATTTCTGC-3′V509Stop5′-CCCTGATTATAGTTCAATGTGAGTTTTACCTACTGAACCAGC-3′5′-GCTGGTTCAGTAGGTAAAACTCACATTGAACTATAATCAGGG-3′1-a The mutated nucleotides are underlined.1-b Coding triplets for the mutants R448K, R448I, R448A, and R448D were AAA, ATC,GCT, and GAC, respectively, in place ofTGA. Open table in a new tab Expression vectors were transformed intoE. coli HMS 174 (DE3), Novagen (Madison, WI). For the expression of N-His tagged MTCYP51, we reduced the temperature to 20 °C after induction with IPTG, which significantly increased the P450 level. Expression of C. albicans and human CYP51 were carried out as described previously (18Stromstedt M. Rozman D. Waterman M.R. Arch. Biochem. Biophys. 1996; 329: 73-81Crossref PubMed Scopus (154) Google Scholar). After expression the cells were pelleted, resuspended (1/10) in 50 mm Tris-HCl (pH 7.8) (containing 1 mm EDTA, 100 in mm NaCl, 0.05% Triton X-100, 0.5 mm phenylmethylsulfonyl fluoride, 10% glycerol, and 0.5 mg/ml lysozyme), incubated for 15 min on ice, homogenized, and then frozen at −70 °C. The total amount of expressed CYP51 was analyzed by size fractionation of E. coli proteins in SDS-polyacrylamide gel. To determine the expression level of P450, cells were thawed and sonicated on ice for 6 × 20 s using a Bronson sonifier (Model 250) at duty cycle 30–40 and 50% maximal output. Insoluble material was removed by centrifugation at 100,000 × g for 20 min. Low expression levels of the MTCYP51 mutants R448K, R448I, and R448A led to modification of previously described (15Bellamine A. Mangla A.T. Nes W.D. Waterman M.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8937-8942Crossref PubMed Scopus (192) Google Scholar) purification conditions. The supernatant after sonication was diluted with 50 mm Tris-HCl (pH 7.4), containing 10% glycerol (buffer A), 40 µmβ-mercaptoethanol and 2 mm imidazole and applied to a Ni2+-nitriloacetic acid-agarose (Qiagen, Valencia, CA), column equilibrated with buffer A containing 20 µmβ-mercaptoethanol and 1 mm imidazole. After extensive three-step washing with buffer A containing 0.1% Triton X-100, buffer A containing 500 mm NaCl and 20 mm Tris-HCl (pH 7.4), containing 10% glycerol (buffer B) and 5 mmimidazole, the P450 was eluted with buffer B containing 50 mm imidazole. The eluate was diluted five times with buffer B and applied onto a Q-Sepharose (Amersham Pharmacia Biotech) column equilibrated with the same buffer. MTCYP51 was eluted with a step gradient of NaCl in buffer B at 0.25 m NaCl. Purified protein was concentrated to 0.2–0.4 mm using Amicon ultrafiltration membrane cones, dialyzed against 50 mmpotassium phosphate (pH 7.4) containing 10% glycerol, and frozen at −70 °C. Absolute and CO difference absorbance spectra were taken using a Beckman DU 640 spectrophotometer. Spectrophotometric indices of the purified low spin proteins were calculated by dividing the Soret peak absorbance (417 nm) by the absorbance at 280 nm. Spin state of the ferric P450 samples was estimated from the ratio (ΔA (393–470)/ΔA (417–470)). The absence of P420 in the purified protein samples was confirmed by CO spectra, which were also used to measure P450 concentrations and kinetics of P450 denaturation of the reduced carbon monoxide complex. Data from four different expression experiments were averaged for MTCYP51 and from eight for C. albicans and human CYP51. Sterol 14α-demethylase activity of wild type and mutant forms of MTCYP51 was determined (15Bellamine A. Mangla A.T. Nes W.D. Waterman M.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8937-8942Crossref PubMed Scopus (192) Google Scholar) using a radiolabeled 24-[3H]DHL/cold DHL mixture as substrate (150,000 cpm/25 nmol in 25 µl of acetone/reaction). Radiolabeled DHL was first purified by HPLC. The molar ratio enzyme/substrate in the reaction mixture was 1/25. E. coli flavodoxin/flavodoxin reductase system served as MTCYP51 electron donor partners (molar ratio 18:2:1). The same mixture with no MTCYP51 was used as a negative control. The final reaction volume was 500 µl and contained 20 mm MOPS (pH 7.4), 50 mm KCl, 5 mmMgCl2, and 10% glycerol. The reaction was stopped by the addition of ethyl acetate. Extracted sterols were dissolved in methanol and analyzed by HPLC using a Walters HPLC equipped with Nova-Pak C18 column (3.9 × 150 mm) and β-RAM radioactivity flow detector (INUS Systems Inc, Tampa, FL). Data were analyzed using Millenium software (15Bellamine A. Mangla A.T. Nes W.D. Waterman M.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8937-8942Crossref PubMed Scopus (192) Google Scholar). CD spectra were recorded on a Jasco J-715 spectropolarimeter (Japan) at 20 °C in thermostated cuvettes with 1-mm optical pathlength for the far UV region and 10-mm pathlength for the visible region. Measurements were conducted in 50 mmpotassium phosphate (pH 7.4) containing 1 and 10 µmMTCYP51 for the far ultraviolet and visible regions, respectively. Buffer with no protein was routinely recorded and subtracted from the original spectra. Data from six scans were averaged. To compare pH-induced denaturation of mutants, 2–5 µl of protein was added to 1 ml of 50 mm phosphate buffer at a set pH, giving a final concentration of 1 µm. For guanidine hydrochloride-induced denaturation, the protein aliquot was added as above to 1 ml of 50 mm potassium phosphate (pH 7.4), containing different concentrations of guanidine hydrochloride, and incubated for 20 min at 20 °C prior to CD measurements at 223 nm. Fluorescence spectra were recorded at room temperature using protein concentration of 1 µm in 50 mm potassium phosphate (pH 7.4), in a 1-cm pathlength cuvette in a SPEX Fluorolog fluorometer. The tryptophan fluorescence was excited at 295 nm with emission spectra being recorded between 320 and 400 nm. In kinetic measurements on pH-induced protein denaturation (pH 5.0), the emission value at 331 nm was plotted as a function of time. Each data point at a given time is the average of four successive traces. Absence of spectrally detectable MTCYP51 upon deletion of the four C-terminal residues (−RTVG) was originally observed for C-His-tagged MTCYP51 (not shown). To exclude any influence of C-terminal histidines on C-terminally mutated proteins, we have changed the position of His tag to the N terminus of MTCYP51. Optimization of expression conditions allowed us to obtain about 550 nmol of wild type MTCYP51/liter of culture (Fig.1A). Deletion of three C-terminal residues (−TVG) does not significantly change P450 expression level, but truncation of the fourth residue (Arg-448) results in no spectrally detectable MTCYP51. Spectrally detected expression levels of P540 correlate well with the results of SDS-PAGE analysis of E. coli soluble fractions (Fig. 1 B). At the same time, the amount of total protein with molecular weight corresponding to MTCYP51 (CYP51 polypeptide chain in inclusion bodies) was not changed, indicating that the truncations neither affect total synthesis nor induce in vivo proteolytic degradation of MTCYP51. Replacement of Arg-448 with lysine, a positively charged residue with shorter side chain, gives about 36% P450 expression relative to wild type. Removal of the positive charge, R448I, decreases expression to 20%. Substitution R448A further reduces side chain length and results in only 5% expressed P450. Mutant R448D containing a negatively charged aspartate at position 448 is not expressed in the P450 form. According to the MTCYP51 crystal structure (16Podust L.M. Poulos T.L. Waterman M.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3068-3073Crossref PubMed Scopus (480) Google Scholar), Arg-448 forms a salt bridge with Asp-287, which is located at the C terminus of the J helix (Fig. 2A). Replacement of Asp-287 with arginine, D287R, decreases the expression of P450 to 13% (Fig. 1 A). Swapping the electrostatic partners in the salt bridge (double mutant D287R/R448D) led to absence of spectrally detectable P450. In addition to the salt bridge with Asp-287, Arg-448 can also bond to a nearby portion of the MTCYP51 molecule. Van der Waals surfaces of its long aliphatic arm tightly adjoin to the surfaces of main chain atoms of residues 412–414, suggesting hydrophobic interactions between them. Dependence of MTCYP51 expression levels on the side chain length of the residue substituted for Arg-448 is in good agreement with this assumption (Fig. 1, Table II). In addition, the crystal structure shows that Arg-448 nitrogens, guanidinium nitrogen Nη1 and amide nitrogen, may form hydrogen bonds with main chain carbonyls of Leu-412 and Glu-414, respectively. Although the hydrogen bond between Nη1 of Arg-448 and carbonyl of Leu-412 is lost upon mutagenesis, the hydrogen bond between main chain atoms is present in all the mutants (Fig.2 B).Table IIPurification and spectral characteristics of MTCYP51 mutantsSampleSide chain length of the 448th residuePurification yield2-aPercentage of yield of detectable P450 in E. coli soluble fraction after two-step purification procedure.Spectrophotometric index2-bSpectrophotometric index 1.6 corresponds to 18 nmol of heme/mg of protein (15).Spin stateÅ%A417/A280ΔA(393–470)/ΔA(417–470)Wild type8.15361.650.41R448K7.07381.700.42R448I4.64381.670.40R448A2.15351.620.422-a Percentage of yield of detectable P450 in E. coli soluble fraction after two-step purification procedure.2-b Spectrophotometric index 1.6 corresponds to 18 nmol of heme/mg of protein (15Bellamine A. Mangla A.T. Nes W.D. Waterman M.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8937-8942Crossref PubMed Scopus (192) Google Scholar). Open table in a new tab Thus, Arg-448 in MTCYP51 forms a link between the C terminus, the J helix, and the C-terminal part of the L helix. This link might be important for 1) maintenance of MTCYP51 structural integrity, 2) heme binding, 3) catalytic activity, or 4) folding of the newly synthesized polypeptide chain. To distinguish between these possibilities, wild type protein and three Arg-448 mutants have been purified and characterized. Because of the strong influence of Arg-448 replacement on P450 expression level, we expected differences in the isolation efficiency of the mutants. However, the yields of pure proteins after the two-stage purification procedure were very similar (Table II). Absolute absorbance spectra of the purified MTCYP51 mutants are shown in Fig. 3A. They all are in the ferric low spin form and have spectrophotometric indexes (A 417/A 280) of approximately 1.6–1.7, suggesting that the mutation does not affect heme insertion. The reduced CO complexes of the mutated P450s show a Soret maximum at the identical position to that of the wild type protein with no detectable P420. Kinetics of denaturation of the CO complexes measured to estimate heme pocket stability show very similar rates of P450 denaturation for wild type and all the mutants (Fig.3 B). Thus, Arg-448 substitution does not affect heme retention. It has been found for human CYP17A that mutation in the C terminus (20Yanase T. Waterman M.R. Zachmann M. Winter J.S. Simpson E.R. Kagimoto M. Biochim. Biophys. Acta. 1992; 1139: 275-279Crossref PubMed Scopus (53) Google Scholar) completely eliminates P450c17 activities. Similar results were obtained for C-terminal truncated CYP2C2 and 2C14 (21Chen C.D. Kemper B. J. Biol. Chem. 1996; 271: 28607-28611Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Enzymatic activities were checked in whole cells without recalculation per the amount of properly folded P450 as has been done in the current work. Based on a CYP17A three-dimensional model, the absence of catalytic activity has been explained by disruption of the structure of β-sheet 3 and mispositioning amino acid residues that form the substrate-binding site (22Auchus R.J. Miller W.L. Mol. Endocrinol. 1999; 13: 1169-1182Crossref PubMed Google Scholar). If this were true for MTCYP51, and Arg-448 were important for maintenance of the local conformation of the substrate-binding site, substitution of Arg-448 must influence catalytic activity of the enzyme. However, all the mutants catalyze DHL 14α-demethylation at about the same rate and efficiency as the wild type (Fig. 3 C). This indicates that Arg-448 is not important for the enzymatic function of MTCYP51. Circular dichroism (CD) spectroscopy is a valuable technique for detecting conformational changes in proteins. Based on changes in CD spectra, it has been shown that C-terminal truncation of staphylococcal nuclease leads to formation of a functionally active intermediate lacking a considerable portion of its helical structure, called a molten globule (23Griko Y.V. Gittis A. Lattman E.E. Privalov P.L. J. Mol. Biol. 1994; 243: 93-99Crossref PubMed Scopus (50) Google Scholar). In adrenodoxin, C-terminal truncation alters the α-helical content and accelerates denaturation (24Bera A.K. Grinberg A. Bernhardt R. Arch. Biochem. Biophys. 1999; 361: 315-322Crossref PubMed Scopus (10) Google Scholar). To evaluate the probability of structural alterations induced by Arg-448 mutation, we measured CD spectra of wild type MTCYP51 and its Arg-448 mutants in the far ultraviolet (UV) and visible regions. Fig. 4A shows the CD spectra in the far UV region (197–250 nm). They are typical for P450 (25Okamoto N. Imai Y. Shoun H. Shiro Y. Biochemistry. 1998; 37: 8839-8847Crossref PubMed Scopus (31) Google Scholar, 26Schulze J. Tschop K. Lehnerer M. Hlavica P. Biochem. Biophys. Res. Commun. 2000; 270: 777-781Crossref PubMed Scopus (10) Google Scholar) and superimposable. The α-helical content estimated from mean residue ellipticity ([θ]R) at 223 nm is close to 50%, which is in a good agreement with 44% determined from the MTCYP51 structure. Thus, Arg-448 mutations do not induce CD-detectable changes in the MTCYP51 secondary structure. CD spectra of the mutants in the visible region are also essentially unchanged (Fig.4 B). Since the CD of P450 in the visible region (negative Cotton effect at 420 nm) reflects proximity of heme to neighboring aromatic residues (27Uchida K. Shimizu T. Makino R. Sakaguchi K. Iizuka T. Ishimura Y. Nozawa T. Hatano M. J. Biol. Chem. 1983; 258: 2512-2519Google Scholar), these data imply that the heme environment is also not influenced by Arg-448 mutation. These results indicate that the mutated proteins lacking salt bridge Asp-287–Arg-448 and hydrophobic interactions with the L helix do not show any alterations in polypeptide backbone structure or heme pocket environment and cannot be classified as functionally active molten globules. We also used CD in the far UV region to compare the equilibrium unfolding behavior of the mutants. For these purposes molar ellipticity at 223 nm, [θ]223, was monitored as a function of pH or guanidine hydrochloride concentration (Fig. 4, C andD). All the samples revealed similar equilibrium unfolding transitions with 50% loss of their α-helical content at pH 5.3 or guanidine hydrochloride concentration of 0.65 m. Lack of noticeable changes in equilibrium unfolding parameters for MTCYP51 mutants indicates the same number of structurally important intramolecular interactions in the mutants as in the wild type. Thus, we conclude that Arg-448 does not contribute to maintenance of MTCYP51 structural integrity. Tryptophan fluorescence is widely used to follow fine changes in protein tertiary structure and stability (23Griko Y.V. Gittis A. Lattman E.E. Privalov P.L. J. Mol. Biol. 1994; 243: 93-99Crossref PubMed Scopus (50) Google Scholar,28van Mierlo C.P. Steensma E. J. Biotechnol. 2000; 79: 281-298Crossref PubMed Scopus (56) Google Scholar). Position of fluorescence emission maxima (330–350 nm) and quantum yield reflect hydrophobicity of the indole ring environment and proximity to tyrosines, which serve as donor for resonance energy transfer. Loss of protein tertiary structure is usually accompanied by red shift of tryptophan emission maximum. In hemoproteins tryptophan fluorescence also strongly depends on the distance from the porphyrin ring, which is a nonradioactive emission acceptor and quenches it. As a result, intrinsic tryptophan fluorescence is
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