Reaction Intermediates in the Catalytic Mechanism of Escherichia coli MutY DNA Glycosylase
2004; Elsevier BV; Volume: 279; Issue: 45 Linguagem: Inglês
10.1074/jbc.m403944200
ISSN1083-351X
AutoresRaymond C. Manuel, Kenichi Hitomi, A.S. Arvai, Paul G. House, Andrew Kurtz, M.L. Dodson, Amanda K. McCullough, John A. Tainer, R. Stephen Lloyd,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoThe Escherichia coli adenine DNA glycosylase, MutY, plays an important role in the maintenance of genomic stability by catalyzing the removal of adenine opposite 8-oxo-7,8-dihydroguanine or guanine in duplex DNA. Although the x-ray crystal structure of the catalytic domain of MutY revealed a mechanism for catalysis of the glycosyl bond, it appeared that several opportunistically positioned lysine side chains could participate in a secondary β-elimination reaction. In this investigation, it is established via site-directed mutagenesis and the determination of a 1.35-Å structure of MutY in complex with adenine that the abasic site (apurinic/apyrimidinic) lyase activity is alternatively regulated by two lysines, Lys142 and Lys20. Analyses of the crystallographic structure also suggest a role for Glu161 in the apurinic/apyrimidinic lyase chemistry. The β-elimination reaction is structurally and chemically uncoupled from the initial glycosyl bond scission, indicating that this reaction occurs as a consequence of active site plasticity and slow dissociation of the product complex. MutY with either the K142A or K20A mutation still catalyzes β and β-δ elimination reactions, and both mutants can be trapped as covalent enzyme-DNA intermediates by chemical reduction. The trapping was observed to occur both pre- and post-phosphodiester bond scission, establishing that both of these intermediates have significant half-lives. Thus, the final spectrum of DNA products generated reflects the outcome of a delicate balance of closely related equilibrium constants. The Escherichia coli adenine DNA glycosylase, MutY, plays an important role in the maintenance of genomic stability by catalyzing the removal of adenine opposite 8-oxo-7,8-dihydroguanine or guanine in duplex DNA. Although the x-ray crystal structure of the catalytic domain of MutY revealed a mechanism for catalysis of the glycosyl bond, it appeared that several opportunistically positioned lysine side chains could participate in a secondary β-elimination reaction. In this investigation, it is established via site-directed mutagenesis and the determination of a 1.35-Å structure of MutY in complex with adenine that the abasic site (apurinic/apyrimidinic) lyase activity is alternatively regulated by two lysines, Lys142 and Lys20. Analyses of the crystallographic structure also suggest a role for Glu161 in the apurinic/apyrimidinic lyase chemistry. The β-elimination reaction is structurally and chemically uncoupled from the initial glycosyl bond scission, indicating that this reaction occurs as a consequence of active site plasticity and slow dissociation of the product complex. MutY with either the K142A or K20A mutation still catalyzes β and β-δ elimination reactions, and both mutants can be trapped as covalent enzyme-DNA intermediates by chemical reduction. The trapping was observed to occur both pre- and post-phosphodiester bond scission, establishing that both of these intermediates have significant half-lives. Thus, the final spectrum of DNA products generated reflects the outcome of a delicate balance of closely related equilibrium constants. Over the last 15 years, multiple laboratories have investigated the catalytic mechanism of DNA glycosylases that initiate the base excision repair (BER) 1The abbreviations used are: BER, base-excision repair; AP, apurinic/apyrimidinic; 8-oxoG, 7,8-dihydro-8-oxoguanine. pathway (reviewed in Refs. 1Stivers J.T. Jiang Y.L. Chem. Rev. 2003; 103: 2729-2759Crossref PubMed Scopus (397) Google Scholar, 2McCullough A.K. Dodson M.L. Lloyd R.S. Annu. Rev. Biochem. 1999; 68: 255-285Crossref PubMed Scopus (333) Google Scholar, 3Dodson M.L. Lloyd R.S. Free Radic. Biol. Med. 2002; 32: 678-682Crossref PubMed Scopus (45) Google Scholar). The elucidation of structure-activity relationships for DNA glycosylases and glycosylase/abasic site (AP) lyases has been facilitated both by solving the crystal structures and cocrystal complexes and analyses of chemical modification and trapping experiments (4Schrock III, R.D. Lloyd R.S. J. Biol. Chem. 1993; 268: 880-886Abstract Full Text PDF PubMed Google Scholar, 5Dodson M.L. Michaels M.L. Lloyd R.S. J. Biol. Chem. 1994; 269: 32709-32712Abstract Full Text PDF PubMed Google Scholar, 6Sun B. Latham K.A. Dodson M.L. Lloyd R.S. J. Biol. Chem. 1995; 270: 19501-19508Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 7Nash H.M. Lu R. Lane W.S. Verdine G.L. Chem. Biol. 1997; 4: 693-702Abstract Full Text PDF PubMed Scopus (162) Google Scholar, 8Morikawa K. Matsumoto O. Tsujimoto M. Katayanagi K. 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Nature. 2000; 403: 859-866Crossref PubMed Scopus (831) Google Scholar, 15Hollis T. Ichikawa Y. Ellenberger T. EMBO J. 2000; 19: 758-766Crossref PubMed Scopus (206) Google Scholar, 16Zharkov D.O. Gilboa R. Yagil I. Kycia J.H. Gerchman S.E. Shoham G. Grollman A.P. Biochemistry. 2000; 39: 14768-14778Crossref PubMed Scopus (34) Google Scholar, 17Fromme J.C. Banerjee A. Huang S.J. Verdine G.L. Nature. 2004; 427: 652-656Crossref PubMed Scopus (271) Google Scholar). These investigations have been successful in both localizing the active site pocket of these enzymes and identifying the key amino acids that participate in the catalytic events that lead to the excision of the inappropriate base. Determination of the chemical steps in the catalytic mechanism of DNA glycosylases is fundamental for understanding how organisms maintain their genome, despite the inevitable damage caused by exogenous and endogenous agents. DNA glycosylases with an associated AP lyase activity (β-elimination) can be distinguished from DNA glycosylases without such activity, based on the identity of the nucleophile that attacks C1′ of the deoxyribose sugar and whether the reaction proceeds through a covalent DNA-enzyme intermediate. DNA glycosylases that also catalyze a β-elimination reaction utilize a primary or secondary amine in the active site, whereas monofunctional glycosylases cleave the glycosyl bond via either the activation of a water molecule or a SN1 attack (reviewed in Refs. 1Stivers J.T. Jiang Y.L. Chem. Rev. 2003; 103: 2729-2759Crossref PubMed Scopus (397) Google Scholar and 18Dodson M.L. Kurtz A.J. Lloyd R.S. Methods Enzymol. 2002; 354: 202-207Crossref PubMed Scopus (7) Google Scholar). When a primary or secondary amine is utilized as the nucleophile, a Schiff base intermediate is formed that can undergo β-elimination, resulting in the cleavage of the phosphodiester bond (5Dodson M.L. Michaels M.L. Lloyd R.S. J. Biol. Chem. 1994; 269: 32709-32712Abstract Full Text PDF PubMed Google Scholar). The identity of the amino acid residue involved in the formation of the Schiff base intermediate can be elucidated by reduction with NaBH4 or NaCNBH3, resulting in the formation of a covalently trapped enzyme-DNA complex that can undergo peptide sequencing. This technique has been widely used as a diagnostic tool to differentiate glycosylases from glycosylase/AP lyases. In recent years, several reports have probed the catalytic mechanism of MutY (12Guan Y. Manuel R.C. Arvai A.S. Parikh S.S. Mol C.D. Miller J.H. Lloyd S. Tainer J.A. Nat. Struct. Biol. 1998; 5: 1058-1064Crossref PubMed Scopus (298) Google Scholar, 16Zharkov D.O. Gilboa R. Yagil I. Kycia J.H. Gerchman S.E. Shoham G. Grollman A.P. Biochemistry. 2000; 39: 14768-14778Crossref PubMed Scopus (34) Google Scholar, 19Zharkov D.O. Grollman A.P. Biochemistry. 1998; 37: 12384-12394Crossref PubMed Scopus (90) Google Scholar, 20Williams S.D. David S.S. Nucleic Acids Res. 1998; 26: 5123-5133Crossref PubMed Scopus (75) Google Scholar, 21Williams S.D. David S.S. Biochemistry. 1999; 38: 15417-15424Crossref PubMed Scopus (42) Google Scholar, 22Williams S.D. David S.S. Biochemistry. 2000; 39: 10098-10109Crossref PubMed Scopus (37) Google Scholar, 23Wright P.M. Yu J. Cillo J. Lu A.L. J. Biol. Chem. 1999; 274: 29011-29018Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 24Chmiel N.H. Golinelli M.P. Francis A.W. David S.S. Nucleic Acids Res. 2001; 29: 553-564Crossref PubMed Scopus (78) Google Scholar, 25Bernards A.S. Miller J.K. Bao K.K. Wong I. J. Biol. Chem. 2002; 277: 20960-20964Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 26Boon E.M. Pope M.A. Williams S.D. David S.S. Barton J.K. Biochemistry. 2002; 41: 8464-8470Crossref PubMed Scopus (33) Google Scholar, 27Boon E.M. Livingston A.L. Chmiel N.H. David S.S. Barton J.K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12543-12547Crossref PubMed Scopus (207) Google Scholar, 28Francis A.W. Helquist S.A. Kool E.T. David S.S. J. Am. Chem. Soc. 2003; 125: 16235-16242Crossref PubMed Scopus (54) Google Scholar); however, this mechanism and an accounting of the roles of catalytic residues have not been formalized. A straightforward interpretation of the complex catalytic mechanism of MutY has remained a challenge due to observations that the enzyme does not demonstrate standard Michaelis-Menten kinetics, with very slow enzyme turnover and apparent redundancies in potential catalytic residues (16Zharkov D.O. Gilboa R. Yagil I. Kycia J.H. Gerchman S.E. Shoham G. Grollman A.P. Biochemistry. 2000; 39: 14768-14778Crossref PubMed Scopus (34) Google Scholar, 19Zharkov D.O. Grollman A.P. Biochemistry. 1998; 37: 12384-12394Crossref PubMed Scopus (90) Google Scholar, 22Williams S.D. David S.S. Biochemistry. 2000; 39: 10098-10109Crossref PubMed Scopus (37) Google Scholar, 24Chmiel N.H. Golinelli M.P. Francis A.W. David S.S. Nucleic Acids Res. 2001; 29: 553-564Crossref PubMed Scopus (78) Google Scholar, 29Porello S.L. Cannon M.J. David S.S. Biochemistry. 1998; 37: 6465-6475Crossref PubMed Scopus (98) Google Scholar). Although there is a consensus on the glycosylase base excision activity of MutY, the AP lyase activity of MutY has remained poorly defined. Biochemical evidence has been presented that argues for MutY being a monofunctional glycosylase (19Zharkov D.O. Grollman A.P. Biochemistry. 1998; 37: 12384-12394Crossref PubMed Scopus (90) Google Scholar, 20Williams S.D. David S.S. Nucleic Acids Res. 1998; 26: 5123-5133Crossref PubMed Scopus (75) Google Scholar, 21Williams S.D. David S.S. Biochemistry. 1999; 38: 15417-15424Crossref PubMed Scopus (42) Google Scholar), whereas other data are consistent with the enzyme being a glycosylase with a concomitant AP lyase activity (30Lu A.L. Yuen D.S. Cillo J. J. Biol. Chem. 1996; 271: 24138-24143Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 31Manuel R.C. Lloyd R.S. Biochemistry. 1997; 36: 11140-11152Crossref PubMed Scopus (78) Google Scholar). Although the latter data suggested an intrinsic AP lyase activity, initial attempts to trap the lyase reaction with NaBH4 were unsuccessful (6Sun B. Latham K.A. Dodson M.L. Lloyd R.S. J. Biol. Chem. 1995; 270: 19501-19508Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Insight into the identity of specific catalytic residues came from the determination of the crystal structure of the 26-kDa catalytic domain of MutY (cMutY, residues Met1-Lys225) complexed with adenine (12Guan Y. Manuel R.C. Arvai A.S. Parikh S.S. Mol C.D. Miller J.H. Lloyd S. Tainer J.A. Nat. Struct. Biol. 1998; 5: 1058-1064Crossref PubMed Scopus (298) Google Scholar). Examination of this structure suggested a reasonable hypothesis to explain and rationalize all prior data. The architecture of the 26-kDa domain of MutY revealed a structure consisting of two subdomains: a 4α-helical, 4Fe-4S cluster domain connected to a 6α-helical bundle containing a helix-hairpin-helix motif (Fig. 1A). Between these subdomains is a deep cleft that binds adenine; the glycosylase active site is composed of two acidic residues Glu37 and Asp138 (shown as the D138N mutant) that are located on opposite sides of the cleft (Fig. 1B). It was hypothesized that these residues have side chains positioned to both protonate N7 of adenine and activate a water molecule for nucleophilic attack at C1′ of the corresponding deoxyribose, a standard glycosylase reaction mechanism. Recently, a catalytically inactive cocrystal complex was solved of a full-length thermophilic homolog of MutY from Bacillus stearothermophilus with DNA containing an A:8-oxoG1 mismatch (17Fromme J.C. Banerjee A. Huang S.J. Verdine G.L. Nature. 2004; 427: 652-656Crossref PubMed Scopus (271) Google Scholar). In this structure, the DNA was bent 55°, and the adenine was fully extruded from the helix and occupied roughly the same position as determined in the previous structure. The reaction mechanism inferred from this structure, suggests that the essential aspartic acid residue stabilizes the positively charged oxocarbenium intermediate in the base cleavage reaction (17Fromme J.C. Banerjee A. Huang S.J. Verdine G.L. Nature. 2004; 427: 652-656Crossref PubMed Scopus (271) Google Scholar). The experimental observation that gave the clue to the source of the lyase reaction came from data that demonstrated that the cMutY bound DNAs containing abasic site analogs with ∼1 nm binding constants. With the enzyme dissociation being very slow, it was hypothesized that a lysine residue (Lys142) would be available for initiating the lyase chemistry through the formation of a Schiff base intermediate (12Guan Y. Manuel R.C. Arvai A.S. Parikh S.S. Mol C.D. Miller J.H. Lloyd S. Tainer J.A. Nat. Struct. Biol. 1998; 5: 1058-1064Crossref PubMed Scopus (298) Google Scholar). Thus, the glycosylase and AP lyase reactions were postulated to be uncoupled, and the lyase chemistry occurred only as a consequence of an opportunistically positioned lysine residue. These hypotheses provided reasonable explanations for why NaBH4-induced trapping of MutY was inefficient, since following the glycosylase reaction, the strong reducing agent NaBH4 will reduce the transiently ring-opened deoxyribose, rendering it unreactive for further β-elimination chemistry. Experimental verification of this hypothesis appeared concomitantly with the crystal structure in which conditions were established for the trapping of a covalent complex between MutY and an A:G mismatch containing DNA with the linkage through Lys142 (16Zharkov D.O. Gilboa R. Yagil I. Kycia J.H. Gerchman S.E. Shoham G. Grollman A.P. Biochemistry. 2000; 39: 14768-14778Crossref PubMed Scopus (34) Google Scholar, 19Zharkov D.O. Grollman A.P. Biochemistry. 1998; 37: 12384-12394Crossref PubMed Scopus (90) Google Scholar, 20Williams S.D. David S.S. Nucleic Acids Res. 1998; 26: 5123-5133Crossref PubMed Scopus (75) Google Scholar, 21Williams S.D. David S.S. Biochemistry. 1999; 38: 15417-15424Crossref PubMed Scopus (42) Google Scholar, 22Williams S.D. David S.S. Biochemistry. 2000; 39: 10098-10109Crossref PubMed Scopus (37) Google Scholar, 23Wright P.M. Yu J. Cillo J. Lu A.L. J. Biol. Chem. 1999; 274: 29011-29018Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 32Zharkov D.O. Rieger R.A. Iden C.R. Grollman A.P. J. Biol. Chem. 1997; 272: 5335-5341Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). However, these conclusions do not rule out the possibility that lysines other than Lys142 could also participate in catalyzing the β-elimination reaction, and none of these data reveal whether the reduction occurs at a pre- or postincision complex. The investigations presented here provide insights into the underlying structural chemistry of the glycosylase and lyase activities of MutY. Materials—All DNA substrates were synthesized by standard phosphoramidite chemistry on a 394 DNA/RNA synthesizer (Applied Biosystems). 8-oxo-7,8-dihydro-2′-deoxyguanine phosphoramidite was purchased from Glen Research. DNA sequencing was performed using an ABI Prism 310 Genetic Analyzer (PerkinElmer Life Sciences). All DNA syntheses and sequence analyses were performed by the NIEHS Center (Molecular Biology Core, Dr. Thomas G. Wood, Director, University of Texas Medical Branch). The plasmid pKKYEco (derivative of pKK223-3; Amersham Biosciences) containing the mutY gene was a gift from Drs. J. H. Miller and M. L. Michaels (UCLA). Adenine, NaBH4, and NaCNBH3 were purchased from Sigma. T4 polynucleotide kinase was purchased from New England Biolabs, Inc., and [γ-32P]ATP was obtained from PerkinElmer Life Sciences. Site-directed Mutagenesis—In vitro site-directed mutagenesis was performed by polymerase chain reaction using the QuikChange™ site-directed mutagenesis kit (Stratagene). Synthetic oligonucleotides used to create the desired changes in the mutY gene were synthesized and purified by gel electrophoresis. Following mutagenesis, the sequence of the entire mutY gene or the region coding for the catalytic domain of MutY (cMutY) was verified by DNA sequence analyses. Amino acids Glu37 and Asp138 were independently changed to cysteines in MutY. In addition, E37S, D138N, K142A, and K20A mutations were engineered independently in cMutY. Protein Purification—Wild type MutY, cMutY, and the mutant forms of these were expressed in Escherichia coli strain CC104mutY-. MutY and cMutY were purified by sequential affinity chromatography (Q-Sepharose, SP-Sepharose, and single-stranded DNA cellulose) as previously described (31Manuel R.C. Lloyd R.S. Biochemistry. 1997; 36: 11140-11152Crossref PubMed Scopus (78) Google Scholar, 33Manuel R.C. Czerwinski E.W. Lloyd R.S. J. Biol. Chem. 1996; 271: 16218-16226Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Similar procedures were adopted for purifying the mutant forms of MutY and cMutY. DNA Substrates—Heteroduplex DNAs used in enzymatic assays are listed in Table I. The oligodeoxynucleotides were purified by electrophoresis through a 15% polyacrylamide gel containing 7 m urea.Table ISequences of synthetic oligonucleotides used as substrate DNAs, where X represents adenine or guanine and Y represents guanine or 8-oxoguanineSequenceSequence 15′-*TACGAATTGCTTAXTTCGTGCAGGCATGGT-3′3′-ATGCTTAACGAATYAAGCACGTCCGTACCA-5′Sequence 25′-*TACGGAGTGAGGCTTGT-3′3′-TGCCTCACGCCGAACAA-5′Sequence 35′-*GGAGTGAGGCTT-3′3′-CCTCACGCCGAA-5′ Open table in a new tab Adenine Glycosylase and AP Lyase Assay—To monitor adenine excision and AP lyase reactions, the DNA strand containing the mismatched adenine (Table I, sequence 1) was radiolabeled at the 5′-end and annealed to the complementary strand containing guanine or 8-oxoG opposite the adenine. The DNA substrate (0.5 nm) was reacted with the enzyme (200 nm) in a reaction buffer containing 25 mm sodium phosphate (pH 6.8), 1 mm EDTA, 50 mm NaCl, and 100 μg/ml bovine serum albumin. The 20-μl reaction mixture was incubated at 37 °C for various times. Aliquots of these reactions were further incubated for 15 min, at 90 °C with piperidine. The reactions were terminated with an equal volume of formamide buffer (95% formamide, 20 mm EDTA, 0.05% xylene cyanol, and 0.05% bromphenol blue) and heated at 90 °C for 4 min prior to separating the DNA reaction products through 15% polyacrylamide gels containing 7 m urea. The substrates and products of the incision reactions were analyzed by PhosphorImager analyses using ImageQuant Software (Amersham Biosciences). Guanine Glycosylase Assays—Duplex DNAs containing G opposite 8-oxoG were used to assay additional mismatch recognition and glycosylase activity (Table I, sequence 1). The strand containing the mismatched guanine was radiolabeled at the 5′-end with [γ-32P]ATP. Two sets of experiments were conducted in which guanine excision was monitored as a function of time or increasing concentrations of MutY. To monitor the kinetics of guanine excision, MutY (400 nm) was incubated with the DNA substrate (2 nm) in a buffer containing 25 mm sodium phosphate (pH 7.0), 1 mm EDTA, 50 mm NaCl, and 100 μg/ml bovine serum albumin. Aliquots were withdrawn at specific times (0, 1, 5, 15, 30, 60, and 300 min) and treated with piperidine for 15 min at 90 °C. Quantitation of guanine glycosylase activity with increasing enzyme concentrations was performed using conditions described above except with different concentrations of MutY (0, 1, 2, 5, 10, 25, 50, and 100 nm). The mismatch cleavage reaction was allowed to proceed for 12 h at 37 °C, prior to the addition of the piperidine. Incubations with piperidine continued for another 15 min before they were terminated and quantitated as described above. Characterization of Schiff Base Intermediates—DNAs containing a single A:G or A:8-oxoG mismatch (Table I, sequences 1-3) were used in the following assay. The strand containing the mismatched adenine was 5′-end-labeled with [γ-32P]ATP. The concentration of each enzyme (MutY, cMutY, cMutY-K142A, and cMutY-K20A) was 200 nm. Substrate DNA (5 nm) and the corresponding protein were incubated in a buffer containing 25 mm HEPES (pH 7.4), 1 mm EDTA, 50 mm NaCl, and 100 μg/ml bovine serum albumin, in the presence of 50 mm NaBH4 or 25 mm NaCNBH3. The 20-μl reaction volume was incubated at 37 °C for 60 min. The reactions were terminated with an equal volume of SDS buffer (50 mm Tris-HCl (pH 6.8), 10 mm dithiothreitol, 2% SDS, 10% glycerol, 0.1% bromphenol blue) and boiled at 100 °C for 5 min. The covalently trapped protein-DNA complexes were resolved through 15% polyacrylamide gels and analyzed by PhosphorImager analyses. Trapping reactions were also performed in which the strand containing the mismatched adenine was 3′-end-labeled with [32P]cordycepin 5′-triphosphate and terminal transferase. DNA substrates were incubated with cMutY or cMutY-K20A in the presence of 50 mm NaBH4. The buffer components, reaction conditions, and analyses of the covalently trapped protein-DNA complexes were similar to that described above for the 5′-end-labeled DNA substrates. Crystallization and Structure Determination of K20A, K142A cMutY, and K20A cMutY-Adenine Complex—Crystals of K20A cMutY were obtained from a solution containing 50 mm Tris-HCl (pH 8.2), 500 mm NaCl, 100 mm MgSO4, 1.2 m Li2SO4, and 10% ethylene glycol. For the K20A cMutY-adenine complex, the mother liquor was saturated with adenine. K142A cMutY was crystallized using a solution containing 100 mm imidazole/malate (pH 8.0), 45% saturated (NH4)2SO4, 500 mm NaCl, and 8% ethylene glycol. To obtain suitable crystals, initial crystals were used for microseeding. Data for K20A and K20A-adenine complex were collected on a Rigaku RU200 rotating anode generator with a Mar image plate detector and beamline 11-1 of the Stanford Synchrotron Radiation Laboratory, respectively, and processed with Denzo and Scalepack (34Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). Data for cMutY K142A was collected at beamline 5.0.2 of the Advanced Light Source and processed with Mosflm and Scala. The structures were solved by molecular replacement using AmoRe with wild type cMutY as a search model (Protein Data Bank number 1MUY) (35Navaza J. Saludjian P. Methods Enzymol. 1997; 276: 581-594Crossref PubMed Scopus (368) Google Scholar). Refinement was done with CNS (36Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar), and manual fitting was done with XFIT (37McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar). Data collection and refinement statistics are summarized in Table II. Coordinates for the three new cMutY structures are deposited in the RCSB Protein Data Bank: accession codes 1WEF (cMutY K20A), 1WEG (cMutY K142A), and 1WEI (cMutY K20A with bound adenine).Table IICrystallographic diffraction data collection and refinementK20AK20A with adenineK142AData collectionBeamlineHomeSSRL 11-1ALSResolution1.91.351.8Wavelength1.540.981Observations174,207476,055182,646Unique reflection19,04251,09325,442Completeness (%)99.2 (93.2)87.5 (57.1)98.1 (88.5)Rsym7.7 (22.9)9.1 (34.1)4.7 (45.2)RefinementResolution20-1.920-1.3525-1.8Reflections18,51341,31521,962R value0.19970.21970.2007Rfree0.22020.23380.2294Root mean square deviation bond lengths0.0080570.00942Root mean square deviation bond angles1.352481.29908Space groupc2c2c2a = 83.7a = 82.7a = 82.9b = 49.4b = 49.4b = 49.0c = 70.0c = 74.1c = 69.7β = 123.2β = 127.6β = 122.8 Open table in a new tab The three new enzyme structures (K20A, K142A, and K20A with adenine) of the 26-kDa catalytic domain of MutY (cMutY) that are reported herein, closely match the x-ray crystal structures of cMutY with either adenine or imidazole bound in the base specificity pocket that were previously solved (Fig. 1A). This experimentally defined preservation of the folds and two-domain structures for these cMutY mutant enzymes suggests that biochemical investigations of these proteins should reflect the functional roles of the mutated residues rather than changes due to misfolding. These new structures and the original structural data are furthermore consistent with localizing the active site of MutY at the interdomain cleft region and suggest putative amino acid residues that contribute to the glycosylase and lyase chemistry of MutY (Fig. 1B) (12Guan Y. Manuel R.C. Arvai A.S. Parikh S.S. Mol C.D. Miller J.H. Lloyd S. Tainer J.A. Nat. Struct. Biol. 1998; 5: 1058-1064Crossref PubMed Scopus (298) Google Scholar). The recent cocrystal structure of a thermophilic homolog of full-length MutY has also confirmed the general positioning of the extrahelical adenine (17Fromme J.C. Banerjee A. Huang S.J. Verdine G.L. Nature. 2004; 427: 652-656Crossref PubMed Scopus (271) Google Scholar). It was hypothesized that Asp138 was the acidic residue that activates a water molecule for nucleophilic attack on the C1′ carbon of the deoxyribose sugar, facilitating the release of the mismatched adenine. Glu37 was hypothesized to protonate N7 of adenine during the nucleophilic displacement reaction. The presence of lyase activity in MutY and the ability of MutY to form Schiff base intermediates led to the hypothesis that an amine group from a side chain of a lysine in the vicinity of the trapped adenine could catalyze the β-elimination reaction. Further analyses of the adenine specificity pocket identified the ϵ-amino group of Lys142 as a candidate residue for the formation of the Schiff base intermediate. Williams and David (21Williams S.D. David S.S. Biochemistry. 1999; 38: 15417-15424Crossref PubMed Scopus (42) Google Scholar) demonstrated that although various lysine mutants of MutY (K16A, K132A, K142A, K157G, and K158A) could nick DNA containing an A:8-oxoG mismatch, only K142A was diminished in the formation of a covalent intermediate in the presence of NaBH4. Additionally, we hypothesized that K20 located within the interdomain loop may modulate the reaction mechanism of MutY (Fig. 1B). The above hypotheses were tested by altering the putative catalytic residues by site-directed mutagenesis and analyzing the wild type and mutant active sites by x-ray crystallography and biochemical enzymology. The detailed atomic structures of cMutY K20A, K142A, and K20A with adenine are identical within experimental error to the cMutY D138N structure reported earlier (12Guan Y. Manuel R.C. Arvai A.S. Parikh S.S. Mol C.D. Miller J.H. Lloyd S. Tainer J.A. Nat. Struct. Biol. 1998; 5: 1058-1064Crossref PubMed Scopus (298) Google Scholar) except for informative local changes and interactions at the mutation site noted here. The crystal structures of cMutY-K20A and cMutY-K142A were determined to 1.9 and 1.8 Å, respectively (Table II, Fig. 2) to provide accurate positions for the mutated residue and its environment. In the wild type structure of cMutY, Lys20 is located on a loop that connects the two subdomains of the [4Fe-4S] cluster and six-helix barrel domain (Fig. 1). A hook, consisting of Lys28 or both Lys20 and Lys28 connects to the [4Fe-4S] domain at the Glu178 main chain through a water molecule, drawing the loop near the two subdomains (Fig. 2A). In D138N and K142A mutant structures, Lys20 twists up 90° toward the surface of the protein, whereas Arg19 turns in the opposite direction into the catalytic pocket (data not shown). The K20A substitution disturbs neither the vicinity of residue 20 nor the active site but is expected to increase flexibility due to the removal of Lys20-forming hydrogen bonds and destabilization of the linker-domain interactions (Fig. 2A). The three-dimensional conservation of the catalytic domain (12Guan Y. Manuel R.C. Arvai A.S. Parikh S.S. Mol C.D. Miller J.H. Lloyd S. Tainer J.A. Nat. Struct. Biol. 1998; 5: 1058-1064Crossref PubMed Scopus (298) Google Scholar) was also used to solve the crystal structure of cMutY in complex with adenine at 1.35 Å by molecular replacement, indicating that the K20A mutant retains the active site features suitable for adenine binding. In this very high resolution structure, the adenine position and interactions are
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