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• W2023361082 abstract "Site-directed mutagenesis has been used to investigate the role of glutamic acid 183 in chloroperoxidase catalysis. Based on the x-ray crystallographic structure of chloroperoxidase, Glu-183 is postulated to function on distal side of the heme prosthetic group as an acid-base catalyst in facilitating the reaction between the peroxidase and hydrogen peroxide with the formation of Compound I. In contrast, the other members of the heme peroxidase family use a histidine residue in this role. Plasmids have now been constructed in which the codon for Glu-183 is replaced with a histidine codon. The mutant recombinant gene has been expressed inAspergillus niger. An analysis of the produced mutant gene shows that the substitution of Glu-183 with a His residue is detrimental to the chlorination and dismutation activity of chloroperoxidase. The activity is reduced by 85 and 50% of wild type activity, respectively. However, quite unexpectedly, the epoxidation activity of the mutant enzyme is significantly enhanced ∼2.5-fold. These results show that Glu-183 is important but not essential for the chlorination activity of chloroperoxidase. It is possible that the increased epoxidation of the mutant enzyme is based on an increase in the hydrophobicity of the active site. Site-directed mutagenesis has been used to investigate the role of glutamic acid 183 in chloroperoxidase catalysis. Based on the x-ray crystallographic structure of chloroperoxidase, Glu-183 is postulated to function on distal side of the heme prosthetic group as an acid-base catalyst in facilitating the reaction between the peroxidase and hydrogen peroxide with the formation of Compound I. In contrast, the other members of the heme peroxidase family use a histidine residue in this role. Plasmids have now been constructed in which the codon for Glu-183 is replaced with a histidine codon. The mutant recombinant gene has been expressed inAspergillus niger. An analysis of the produced mutant gene shows that the substitution of Glu-183 with a His residue is detrimental to the chlorination and dismutation activity of chloroperoxidase. The activity is reduced by 85 and 50% of wild type activity, respectively. However, quite unexpectedly, the epoxidation activity of the mutant enzyme is significantly enhanced ∼2.5-fold. These results show that Glu-183 is important but not essential for the chlorination activity of chloroperoxidase. It is possible that the increased epoxidation of the mutant enzyme is based on an increase in the hydrophobicity of the active site. Chloroperoxidase (CPO 1The abbreviations used are: CPOchloroperoxidaseIEFisoelectric focusingALAδ-aminolevulinic acid; EC 1.11.1.10) is a heavily glycocylated heme peroxidase secreted from the filamentous fungus Caldariomyces fumago (1Morris D.R. Hager L.P. J. Biol. Chem. 1966; 241: 1763-1768Abstract Full Text PDF PubMed Google Scholar, 2Shaw P.D. Hager L.P. J. Biol. Chem. 1961; 236: 1626-1630Abstract Full Text PDF Google Scholar, 3Thomas J.A. Morris D.R. Hager L.P. J. Biol. Chem. 1970; 245: 3135-3142Abstract Full Text PDF PubMed Google Scholar). Natural product research in the 1940s discovered the biosynthesis of chlorinated products by C. fumago (4Clutterbuck P.W. Mukhopadhyay S.L. Oxford A.E. Raistrick H. Biochem. J. 1940; 34: 664-677Crossref PubMed Google Scholar). Caldariomycin (2,2-dichloro-1,3-cyclopentanediol) is the major chlorinated compound found in C. fumago cultures. Much later, enzyme and tracer experiments delineated the role of CPO in the biosynthesis of caldariomycin (5Beckwith J.R. Clark R. Hager L.P. J. Biol. Chem. 1963; 238: 3086-3090Abstract Full Text PDF PubMed Google Scholar, 6Beckwith J.R. Hager L.P. J. Biol. Chem. 1963; 238: 3091-3095Abstract Full Text PDF PubMed Google Scholar). CPO is undoubtedly the most versatile member of the heme peroxidase family. In addition to catalyzing chlorination, bromination, and iodination reactions (7Hager L.P. Morris D.R. Brown F.S. Eberwein H. J. Biol. Chem. 1966; 241: 1769-1777Abstract Full Text PDF PubMed Google Scholar, 8Thomas J.A. Hager L.P. Biochem. Biophys. Res. Commun. 1969; 35: 444-450Crossref PubMed Scopus (26) Google Scholar, 9Thomas J.A. Morris D.R. Hager L.P. J. Biol. Chem. 1970; 245: 3129-3134Abstract Full Text PDF PubMed Google Scholar), CPO catalyzes typical one electron oxidations (10Hewson W.D. Hager L.P. J. Phycol. 1980; 16: 340-345Crossref Scopus (79) Google Scholar) and possesses catalase (11Hager, L. P., Doubek, D. L., Silverstein, R. M., Lee, T. T., Thomas, J. A., Hargis, J. H., Martin, J. C., Oxidase Related Redox System, Proceedings of International Symposium, 2nd Ed., King, T. E., Mason, H. S., Morrison, M., 1, 1973, 311, 332, University Park Press, Baltimore, MD.Google Scholar) and monooxygenase activities (12Hollenberg P. FASEB J. 1992; 2: 686-694Crossref Scopus (96) Google Scholar). CPO is especially adept in catalyzing a number of chiral oxidations. The chiral epoxidation of olefins (13Allain E.J. Hager L.P. Deng L. Jacobsen E.N. J. Am. Chem. Soc. 1993; 115: 4415-4416Crossref Scopus (230) Google Scholar, 14Dexter A.F. Hager L.P. J. Am. Chem. Soc. 1995; 117: 817-818Crossref Scopus (54) Google Scholar, 15Lakner F.J. Hager L.P. J. Org. Chem. 1996; 61: 3923-3925Crossref PubMed Scopus (95) Google Scholar, 16Lakner F.J. Cain K.P. Hager L.P. J. Am. Chem. Soc. 1997; 119: 443-444Crossref Scopus (70) Google Scholar), sulfides (17Doerge D.R. Arch. Biochem. Biophys. 1986; 244: 678-685Crossref PubMed Scopus (61) Google Scholar, 18Colonna S. Gaggero N. Manfredi A. Casella L. Gullotti M. Carrea G. Pasta P. Biochemistry. 1990; 29: 10465-10468Crossref PubMed Scopus (153) Google Scholar, 19Alvarez J.C. Ortiz de Montellano P.R. Biochemistry. 1992; 31: 8315-8322Crossref PubMed Scopus (30) Google Scholar), and indole (20Corbett M.D. Chipko B.R. Biochem. J. 1970; 183: 269-276Crossref Scopus (55) Google Scholar, 21van Deurzen M.P.J. van Rantwijk F. Sheldon R.A. J. Mol. Catal. B: Enzymol. 1996; 2: 33-42Crossref Scopus (63) Google Scholar, 22Miller V.P. Tschirret-Guth R.A. Ortiz de Montellano P.R. Arch. Biochem. Biophys. 1995; 319: 333-341Crossref PubMed Scopus (77) Google Scholar) are carried out in high yield and enantioselectivity. CPO also catalyzes chiral benzylic (23Zaks A. Dodds D.R. J. Am. Chem. Soc. 1995; 117: 10419-19424Crossref Scopus (20) Google Scholar) and propargylic hydroxylations (24Hu S. Hager L.P. Biochem. Biophys. Res. Commun. 1998; 253: 544-546Crossref PubMed Scopus (28) Google Scholar, 25Hu S. Hager L.P. J. Am. Chem. Soc. 1999; 121: 872-873Crossref Scopus (89) Google Scholar). The stereoselective abilities of CPO in chiral catalysis suggest that native CPO and/or engineered CPO mutants have the potential to become important industrial catalysts. chloroperoxidase isoelectric focusing δ-aminolevulinic acid The initial reaction of CPO with hydrogen peroxide generates an oxidized intermediate, Compound I. This oxidized intermediate contains one oxygen atom and both of the oxidizing equivalents originally present in hydrogen peroxide. One of the oxidizing equivalents is associated with the conversion of heme ferric iron to an oxyferryl iron species, and the other is generated by the removal of one of the electrons from the heme porphyrin, generating a porphyrin π cation radical (26Roberts J.E. Hoffman B.M. Rutter R.J. Hager L.P. J. Biol. Chem. 1981; 256: 2118-2121Abstract Full Text PDF PubMed Google Scholar, 27Rutter R.J. Hager L.P. J. Biol. Chem. 1999; 257: 7958-7961Abstract Full Text PDF Google Scholar). It is assumed that the versatile functions of CPO must be based on a CPO unique active site structure that includes a proximal thiolate ligand to the heme iron and a glutamic acid residue distal to the heme (28Sundaramoorthy M. Terner J. Poulos T.L. Structure. 1995; 3: 1367-1377Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). It has been proposed that Glu-183 functions as a general acid-base catalyst in facilitating the formation of Compound I. In contrast, most if not all other peroxidases have histidines functioning in this role. In previous experiments, the proximal thiolate ligand (Cys-29) has been replaced with a histidine residue and the mutant enzyme was expressed in C. fumago (29Yi X. Mroczko K.M. Wang X. Hager L.P. Proc. Natl. Acad. Sci. U. S. A. 1999; (12412–12417)PubMed Google Scholar). In this mutant, thekcat for chlorination and peroxidation was reduced ∼95%. The catalase and epoxidation activity in the mutant were reduced ∼70%. It is obvious that cysteine 29 plays an important role in CPO catalysis. However, unlike the comparable cytochrome P450 Cys to His mutant, the replacement of CPO cysteine 29 with histidine does not totally abolish CPO activities. The distal Glu-183 also plays an important role in the catalytic reactions of CPO. Attempts to express a Glu-183 to His mutant in C. fumago were unsuccessful probably because the mutation is a lethal event. Previous attempts to produce CPO knock-outs in C. fumago also systematically failed. Because it is highly unlikely that CPO, a secreted enzyme, is an essential gene product in C. fumago, there must be an alternate explanation for these failures. The opposite strand DNA to the DNA coding for CPO contains an open reading frame; thus, it is quite possible that CPO mutations could prove to be lethal by producing a mutation in the opposite strand DNA. Recently, Conesaet al. (30Conesa A. van de Velde F. van Rantwijk F. Sheldon R.A. Punt P.J. van den Hondel C.A. J. Biol. Chem. 2001; 276: 17635-17640Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) have shown that the wild typeCPO gene can be expressed in Aspergillus nigerwith the recombinant protein retaining the catalytic properties of the native C. fumago enzyme. Therefore, this expression system was chosen for the production of the Glu-183 to His mutant. This paper describes the preparation, expression, properties, and catalytic activities of this CPO mutant. Restriction enzymes were purchased from New England Biolabs (Beverly, MA) and Invitrogen. T4 DNA ligase was purchased fromPromega (Madison, WI). Purified oligonucleotides were purchased from Keck Biotechnique Center at University of Illinois. Unless otherwise noted, chemicals were obtained from commercial sources and used without further purification. Mutations were carried out using the QuikChange site-directed mutagenesis kit of Stratagene (La Jolla, CA). The mutations were introduced via PCR amplification of pTZC using synthetic oligonucleotide primers. The primers contained a silent mutation site that could be used in a convenient mutant-screening assay. Primers for site-directed mutagenesis were designed with the aid of the Wisconsin GCG Package, version 9.1 (Madison, WI). PCR reactions were carried out in a PerkinElmer thermal cycler (PerkinElmer Life Sciences). Standard “hot-start” PCR was performed using high fidelity pfupolymerase. The PCR products were extracted and purified from agarose gels using kits purchased from Qiagen (Valencia, CA). The desired mutations were identified by restriction analysis and confirmed by DNA sequencing. Expression vector pgpd/CPOE183H was constructed via a three-way ligation. The three DNA sequences used in the construction were: 1) a 230-bp AflIII/XmnI fragment derived from pCPO3 (30Conesa A. van de Velde F. van Rantwijk F. Sheldon R.A. Punt P.J. van den Hondel C.A. J. Biol. Chem. 2001; 276: 17635-17640Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) that contained the N-terminal CPO coding sequence, 2) a ∼900-bpXmnI/BamHI fragment derived from pTZC (31Mead D.A. Szczesna-Skorupa E. Kemper B. Protein Eng. 1986; 1: 67-74Crossref PubMed Scopus (569) Google Scholar) that contained the remaining CPO coding sequence, and 3) an A. niger expression vector, pAN52–5Not (GenBankTMaccession number Z32750), that contained an A. nidulans gpdpromoter and the A. niger trpC terminator sequences (32Conesa A. van den Hondel C.A. Punt P.J. Appl. Environ. Microbiol. 2000; 66: 3016-3023Crossref PubMed Scopus (111) Google Scholar). The XmnI/BamHI fragment contained the E183H mutation. The A. niger vector was digested withNcoI and BamHI to create an insertion site for the CPO sequences (Fig. 1). Plasmid gpd/CPOE183H was co-transformed with pAB4-1 in A. niger MGG029 according to previously described procedures (33Conesa A. Weelink G. van den Hondel C.A. Punt P.J. FEBS Lett. 2001; 503: 117-120Crossref PubMed Scopus (12) Google Scholar). Transformants were screened in situ by immunodetection as described previously (34Zong, Q., Expression of Recombinant Chloroperoxidase.Ph.D. thesis, 1997, University of Illinois.Google Scholar). The mutant clones were grown in roller bottles at 22 °C for 5–7 days. Each bottle contained 300 ml of 5% maltose, 5% yeast extract, 0.5% casein amino acids, 50 μm hemin (Sigma), and 100 μm δ-aminolevulinic acid (Sigma) and the Hutner metal ion supplement (35Hutner S.H. Provosoli L. Schatz A. Haskins C.P. Proc. Am. Phil. Soc. 1950; 94: 152-153Google Scholar). To stabilize the mutant chlorination and epoxidations activities, all of the purification buffer solutions contained 50 μm hemin. Crude culture medium containing the secreted enzyme (1.5 liters) was concentrated 50-fold and applied to a DEAE-Sepharose CL-6B ion-exchange chromatography column (4 × 30 cm) that had been equilibrated with 50 mm Bis/Tris at pH 6.5. The flow rate was adjusted 1 ml/min. Chromatography was carried out at 4 °C. The protein was eluted from the column with a step gradient of 50 mmBis/Tris buffer at pH 6.5 containing 10–70 mm ammonium sulfate. Each fraction was assayed for chlorination activity to identify the active fractions. The active fractions appeared in the eluent fractions containing 28 mm ammonium sulfate. The enzyme was concentrated by ultrafiltration (Amicon, Newburyport, MA). The purity of the mutant enzyme was examined by 12.5% SDS-PAGE gel (Invitrogen) under denaturing condition (36Laemmli U. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207530) Google Scholar). The enzymes were visualized by staining with Coomassie Brilliant Blue R-250. IEF electrophoresis was performed using the Pharmacia Phastsystem with IEF PhastGels (pH 3–9) and the Pharmacia IEF Mix 2.8–6.5 kit. IEF gels were visualized by silver staining. Heme proteins were quantitatively determined using the hemochromogen analysis developed by Deeb and Hager (37Deeb S.S. Hager L.P. J. Biol. Chem. 1964; 239: 1024-1031Abstract Full Text PDF PubMed Google Scholar). This information provided a basis for comparing the relative enzymatic activities of the mutant and native CPO. Because both apomutant and holomutant enzymes were secreted in the A. niger cultures, the total concentration of mutant enzyme was measured in a dot blot assay. Aliquots of the partially purified mutant enzyme or aliquots of the crude culture medium were loaded onto nitrocellulose membranes. The membrane was incubated with anti-CPO rabbit polyclonal IgG antibody for 1 h at room temperature. After incubation, the membrane was washed and then incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G for 1 h. After this second incubation step, the membrane was washed and then reacted with diaminobenzidine and hydrogen peroxide for 10 min for color development. The total concentration of apomutant plus holomutant enzyme was estimated by comparing the dot blot results with the results obtained from serial dilutions of known concentrations of native CPO. Far-UV-CD spectra were recorded on a JASCO J-720 spectropolarimeter at room temperature. Two spectra were recorded from 250 to 200 nm at 0.1-nm intervals with a spectral bandwidth of 1 nm (the spectra were averaged). The concentration of partially purified samples for both the mutant and native CPO control was identical (50 μg/ml). The relation between activity and pH was examined from pH 2.5 to 7.0 using the potassium phosphate buffers. All UV-visible absorption measurements were determined in a Shimadzu UV-1201 spectrophotometer using 1-cm path length quartz cuvettes. The chlorination of monochlorodimedone to dichlorodimedone was used for measuring the chlorination activity of the mutant CPO (7Hager L.P. Morris D.R. Brown F.S. Eberwein H. J. Biol. Chem. 1966; 241: 1769-1777Abstract Full Text PDF PubMed Google Scholar). The reaction mixture contained 20 mm potassium phosphate buffer, pH 2.75, 20 mm KCl, 0.5 mm monochlorodimedone, 2 mm H2O2, and a suitable aliquot of the enzyme in a total volume of 3 ml. The reaction was initiated by the addition of enzyme, and the decrease in absorbance at 278 nm was monitored at room temperature. One unit of chlorination activity was defined as the formation of 1 μmol of dichlorodimedone per second. The epoxidation ofp-nitrostyrene was used to measure epoxidation activity according to the procedure developed by Rai et al. (38Rai G.P. Sakai S. Florez A.M. Mogollon L. Hager L.P. Adv. Synth. Catal. 2001; 343: 638-645Crossref Google Scholar). The reaction mixture contained 100 mm sodium acetate buffer, pH 4.5, 0.2 mmp-nitrostyrene, and 2 mmH2O2 plus a suitable aliquot of the enzyme in a total volume of 3 ml. The activity was determined by monitoring the decrease in absorbance at 312 nm as a function of time. One unit of epoxidation activity was defined as the formation of 1 μmol ofp-nitrostyrene oxide per second. The oxidation of reduced methylene blue was used to measure catalase activity (38Rai G.P. Sakai S. Florez A.M. Mogollon L. Hager L.P. Adv. Synth. Catal. 2001; 343: 638-645Crossref Google Scholar). The reaction mixture contained 100 mm sodium acetate buffer, pH 4.5, 0.2 mm dithionite reduced methylene blue, 2 mmH2O2, and a suitable aliquot of enzyme in a total volume of 3 ml. One unit of catalase activity was defined as the formation of 1 μmol of the oxidized form of methylene blue per second. A. niger produces the E183H mutant enzyme at levels comparable to the expression of wild type CPO in A. niger. However, a majority of the mutant CPO expressed in A. niger is in the form of apoenzyme having no heme incorporated. By comparing the relative levels of apomutant and holomutant enzymes estimated from a hemochromogen assay and a dot blot immunoassay, the ratio of apoenzyme to holoprotein was ∼1.5 to 1. A similar observation has been made in the expression of recombinant wild type CPO in A. niger (30Conesa A. van de Velde F. van Rantwijk F. Sheldon R.A. Punt P.J. van den Hondel C.A. J. Biol. Chem. 2001; 276: 17635-17640Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Most disturbingly, the E183H presented serious problems in early attempts to purify and characterize the mutant enzyme. In early studies, the Glu-183 mutant lost most of its chlorination and epoxidation activity in simple dialysis and ultrafiltration steps. Acetone precipitation, a regular purification step used with native CPO, could not be used because the mutant lost almost all activity when exposed to acetone at −20 °C. Subsequently, it was discovered that the loss of the chlorination and epoxidation activity during dialysis and ultrafiltration could be prevented by the addition of iron protoporyphyrin IX to the mutant enzyme preparations. The presence of heme greatly stabilized the E183H extracts, and in addition, 90% of the mutant protein now appeared in the form of holoenzyme. In contrast, holoenzyme represented only one-third of the mutant enzyme population in untreated extracts. Conesa et al. (30Conesa A. van de Velde F. van Rantwijk F. Sheldon R.A. Punt P.J. van den Hondel C.A. J. Biol. Chem. 2001; 276: 17635-17640Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) found that secreted wild type CPO inA. niger was subject to partial degradation by secreted protease activity. In our initial problems concerning the instability of the E183H mutant enzyme, we considered protease degradation as a potential cause of the instability. This turned out not to be true. Ten different protease inhibitors either singly or in kit form were tested and were found to have no effect. The inhibitors tested were antipain-dihydrochloride, bestatin, chymotatin, E-64, leupeptin, pepstatin, phosphoramidon, pefablioc SC, aprotinin, and phenylmethylsulfonyl fluoride. Other reagents such as bovine serum albumin, glycerol, and sorbitol had no effect on the stability of the E183H enzyme. In regular culture broth, the secretion level of the mutant protein (apoenzyme plus holoenzyme) was quite low (∼2 mg/liter). The addition of ALA to the culture medium increased the secretion level ∼4-fold. ALA synthase is thought to be a crucial enzyme to regulate heme synthesis. There are a few reports regarding an increase in heme protein production by ALA inducement. For example, Herbaud et al. (39Herbaud M.L. Aubert C. Durand M.C. Guerlesquin F. Thony-Meyer L. Dolla A. Biochim. Biophys. Acta. 2000; 1481: 18-24Crossref PubMed Scopus (34) Google Scholar) found that the highest cytochrome c3 production was obtained whenEscherichia coli was grown in LB medium supplemented with δ-aminolevulinic acid under aerobic condition (39Herbaud M.L. Aubert C. Durand M.C. Guerlesquin F. Thony-Meyer L. Dolla A. Biochim. Biophys. Acta. 2000; 1481: 18-24Crossref PubMed Scopus (34) Google Scholar). Further studies showed that the regulative effects of ALA and hemin on ALA synthase were done through transcriptional regulation (40Ades I.Z. Stevens T.M. Drew P.D. Arch. Biochem. Biophys. 1987; 253: 297-304Crossref PubMed Scopus (24) Google Scholar). In these studies on the production of mutant CPO by A. niger, the addition of δ-aminolevulinic acid significantly enhanced the production of the E183H mutant enzyme. The instability of the mutant enzyme precluded the normal initial acetone precipitation routinely used to purify native CPO. Even the presence of heme did not protect Glu-183 from denaturation by acetone. DEAE ion exchange chromatography was used exclusively for purification of the mutant enzyme. As described under “Experimental Procedures,” all purification buffers contained 50 μm of hemin to stabilize the mutant enzyme. The specific activity of the partially purified E183H increased by 145-fold, and the enzyme showed a major band in SDS-PAGE (Fig. 2) and had a RZ value of 1.2 (Fig. 3).Figure 3Absorption spectrum of the partially purified E183H. The RZ value, ratio ofA400 to A280, is 1.2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To evaluate the effect of the distal substitution on the various activities of CPO, the activities of the E183H mutant were measured in three different assays. The chlorination activity of the mutant enzyme decreased to a level of ∼15% of the wild type activity, and the catalase activity decreased to 50% of the native CPO catalytic activity (Fig. 4). As outlined under “Experimental Procedures,” the epoxidation ofp-nitrostyrene was used to assay the epoxidation activity of the E183H mutant according to the procedure developed by Rai et al. (38Rai G.P. Sakai S. Florez A.M. Mogollon L. Hager L.P. Adv. Synth. Catal. 2001; 343: 638-645Crossref Google Scholar). In contrast to the results obtained for the chlorination and dismutation mutant activities, epoxidation activity unexpectedly increased 2.5-fold (Fig. 4). To further elucidate the role of Glu-183 as a general acid-base catalysis, the pH optimum of the E183H mutant was examined. The enzyme activity was measured at various pH values ranging from pH 2.5 to 7.0. The concentration of H2O2 used in these studies was optimized for each pH value. The mutant was most active at pH 3.0, whereas its native counterpart was at pH 2.75 (Fig. 5). The E183H mutant showed a slightly higher pI value based on their isoelectric focusing gel properties (Fig.6), but this was expected for the replacement of a negatively charged by a positively charged one.Figure 6pI of the E183H mutant. This figure illustrates IEF using a PhastGel (pH 3–9) for the E183H mutant and native CPO, which shows that the pI of the mutant is slightly higher than that of native CPO. Lane 1, markers; lane 2, mutant CPO; lane 3, native CPO.View Large Image Figure ViewerDownload Hi-res image Download (PPT) CD spectrum was used to exclude the possibility that activities of mutant enzyme were decreased by large structure changes. The CD data showed that there were some small differences in intensity in the peptide bond region between the mutant and native CPO; however, the overall shapes of the two spectra were the same (Fig. 7). The CD spectra of the mutant proteins (80.5% α-helix, 6.2% β-turn, and 15.7% unordered) were very similar to that of wild type (79.6% α-helix, 0.6% β-sheet, 6.4% β-turn, and 16.2% unordered). The result indicated that the amino acid substitutions had no significant effect on the overall folding of the mutant enzyme. The discovery that hemin protects the mutant enzyme during purification and the observation that the apoform of the mutant can be converted to the holoenzyme in the presence of hemin were extremely important observations because hemin allowed the purification and characterization of this mutant enzyme. This discovery may also offer a new approach to the production of mutant CPOs. Wild type apoCPO can be produced in E. coli in large quantities; however, under the very best conditions, only small amounts of holoenzyme could be obtained by reconstitution procedures (41Zong Q. Osmulski P.A. Hager L.P. Biochemistry. 1995; 34: 12420-12425Crossref PubMed Scopus (23) Google Scholar). Because the E183H apomutant readily reconstitutes to holoenzyme and because the E183H mutant is more active than wild type CPO in chiral epoxidation catalysis, it appears logical to assume that further mutation of E183H in E. coli is an obvious choice for examining the active site requirements for chiral catalysis. Histidine plays a key role as a general acid-base catalyst in the formation of Compound I in the plant peroxidases. Poulos and Kraut (42Poulos T.L. Kraut J. J. Biol. Chem. 1980; 255: 10322-10330Abstract Full Text PDF PubMed Google Scholar) proposes that the distal histidine assists the peroxide anion to bind the heme iron by acting as a general base catalyst while accepting a proton from the neutral hydrogen peroxide when it binds to the iron and by acting as a general acid catalyst by donating a proton to the departing hydroxide anion (42Poulos T.L. Kraut J. J. Biol. Chem. 1980; 255: 10322-10330Abstract Full Text PDF PubMed Google Scholar). Several mutant peroxidases have revealed histidine as an important active site residue. In wild type CPO, a glutamic acid residue replaces the plant peroxidase histidine distal residue and is postulated to function as the acid-base catalyst, facilitating the formation of Compound I. By analogy to the experiments with horseradish peroxidase where replacement of the distal histidine leads to large losses in activity (43Erman J.E. Vitello L.B. Miller M.A. Shaw A. Brown K.A. Kraut J. Biochemistry. 1993; 32: 9798-9806Crossref PubMed Scopus (186) Google Scholar), it would be expected that replacement of the distal glutamic acid in CPO also should be deleterious. This is certainly true in terms of the halogenation activity of CPO. The rate of enzymatic chlorination in the Glu-183 to His mutant falls by ∼85%. This indicates that the distal Glu residue in native CPO is important but not essential for the halogenation activity of the enzyme. The van der Waals volume of histidine (98.8 cm3 mol−1) is larger than that of histidine (85.9 cm3 mol−1), so some distortion of the mutant active site should be expected. The fact that the mutant heme prosthetic group is more loosely held supports the conclusion that there has been a change in the geometry of the active site. The x-ray structure of native CPO identifies a proton shuttle among Glu-183, His-105, and Asp-106 (44Sundaramoorthy M. Mauro J.M. Sullivan A.M. Terner J. Poulos T.L. Acta Crystallogr. Sec. D. 1995; 51: 842-844Crossref PubMed Scopus (19) Google Scholar). When Glu-183 is replaced with a histidine residue, the normal orientation of this triad could be partially destroyed. Such a change could obviously impair the chlorination activity of the mutant. In studies with horseradish peroxidase mutants, it has been shown that the loss of a hydrogen bond between the distal His-42 and Asp-70 induces severe functional defects in peroxidase activity (45Tanaka M. Ishimori K. Mukai M. Kitagawa T. Morishima I. Biochemistry. 1997; 36: 9889-9898Crossref PubMed Scopus (76) Google Scholar). In addition, previous experiments have shown that the modification of a single histidine residue with diethyl pyrocarbonate totally inactivates CPO with respect to chlorination activity (46Blanke S.R. Hager L.P. J. Biol. Chem. 1990; 265: 12454-12461Abstract Full Text PDF PubMed Google Scholar). A quite different picture emerges from a comparison of the catalytic activity of the mutant and native CPOs in the epoxidation reaction. In this case, the mutant is ∼2.5-fold more active than the native enzyme. In focusing on the origin of this increase in activity, it is highly unlikely that the increase in activity could be associated with the formation of Compound I, the first step in all CPO oxidations. The rate constant for the formation of Compound I in the reaction of native enzyme with hydrogen peroxide is 2.4 × 106m−1sec−1 (47Araiso T. Rutter R. Palcic M.M. Hager L.P. Dunford H.B. Can. J. Biochem. 1981; 59: 233-236Crossref PubMed Scopus (35) Google Scholar), whereas the rate constants for both the chlorination and epoxidation activity of native CPO are magnitudes lower. Thus, the overall rate for both chlorination and epoxidation reactions must reside in the second step involving the reaction between Compound I and the oxidizable substrate. It seems probable that the distortion of the active site that inhibits the chlorination activity must actually facilitate the reaction between the olefinic substrate and the oxygen atom in the oxyferryl Compound I species, perhaps by allowing a closer approach of the olefinic substrate to the oxygen atom of the oxyferryl group in Compound I. Clearly, the olefinic substrate must approach the Compound I intermediate from the distal side of the heme. In the mechanism-based suicide reaction between CPO Compound I andcis-β-methyl styrene, an oxygen-carbon bond is formed in the inactive intermediate (48Debrunner P.G. Dexter A.F. Xia Y.-M. Hager L.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12791-12798Crossref PubMed Scopus (22) Google Scholar). In a similar vein, the proton shuttle among Glu-183, His-105, and Asp-106 that is important for chlorination activity appears unimportant for epoxidation activity. The diethyl pyrocarbonate treatment that kills chlorination activity does not impair epoxidation activity. We thank Dr. Shaomin Tian for CD data processing." @default.
• W2023361082 created "2016-06-24" @default.
• W2023361082 creator A5002377126 @default.
• W2023361082 creator A5032557742 @default.
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• W2023361082 date "2003-04-01" @default.
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• W2023361082 title "Examining the Role of Glutamic Acid 183 in Chloroperoxidase Catalysis" @default.
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