Structures of Liganded and Unliganded RsrI N6-Adenine DNA Methyltransferase

Abstract

The structures of RsrI DNA methyltransferase (M.RsrI) bound to the substrate S-adenosyl-l-methionine (AdoMet), the product S-adenosyl-l-homocysteine (AdoHcy), the inhibitor sinefungin, as well as a mutant apo-enzyme have been determined by x-ray crystallography. Two distinct binding configurations were observed for the three ligands. The substrate AdoMet adopts a bent shape that directs the activated methyl group toward the active site near the catalytic DPPY motif. The product AdoHcy and the competitive inhibitor sinefungin bind with a straight conformation in which the amino acid moiety occupies a position near the activated methyl group in the AdoMet complex. Analysis of ligand binding in comparison with other DNA methyltransferases reveals a small, common subset of available conformations for the ligand. The structures of M.RsrI with the non-substrate ligands contained a bound chloride ion in the AdoMet carboxylate-binding pocket, explaining its inhibition by chloride salts. The L72P mutant of M.RsrI is the first DNA methyltransferase structure without bound ligand. With respect to the wild-type protein, it had a larger ligand-binding pocket and displayed movement of a loop (223–227) that is responsible for binding the ligand, which may account for the weaker affinity of the L72P mutant for AdoMet. These studies show the subtle changes in the tight specific interactions of substrate, product, and an inhibitor with M.RsrI and help explain how each displays its unique effect on the activity of the enzyme. The structures of RsrI DNA methyltransferase (M.RsrI) bound to the substrate S-adenosyl-l-methionine (AdoMet), the product S-adenosyl-l-homocysteine (AdoHcy), the inhibitor sinefungin, as well as a mutant apo-enzyme have been determined by x-ray crystallography. Two distinct binding configurations were observed for the three ligands. The substrate AdoMet adopts a bent shape that directs the activated methyl group toward the active site near the catalytic DPPY motif. The product AdoHcy and the competitive inhibitor sinefungin bind with a straight conformation in which the amino acid moiety occupies a position near the activated methyl group in the AdoMet complex. Analysis of ligand binding in comparison with other DNA methyltransferases reveals a small, common subset of available conformations for the ligand. The structures of M.RsrI with the non-substrate ligands contained a bound chloride ion in the AdoMet carboxylate-binding pocket, explaining its inhibition by chloride salts. The L72P mutant of M.RsrI is the first DNA methyltransferase structure without bound ligand. With respect to the wild-type protein, it had a larger ligand-binding pocket and displayed movement of a loop (223–227) that is responsible for binding the ligand, which may account for the weaker affinity of the L72P mutant for AdoMet. These studies show the subtle changes in the tight specific interactions of substrate, product, and an inhibitor with M.RsrI and help explain how each displays its unique effect on the activity of the enzyme. Biological DNA methylation is ubiquitous and important in many cellular mechanisms including genetic imprinting, immunity, cancer, and gene regulation (1Paulsen M. Ferguson-Smith A. J. Pathol. 2001; 195: 97-110Crossref PubMed Scopus (218) Google Scholar, 2Low D. Weyand N. Mahan D. Infect. Immun. 2001; 69: 7197-7204Crossref PubMed Scopus (256) Google Scholar, 3Jeltsch A. ChemBioChem. 2002; 3: 274-293Crossref PubMed Google Scholar). In bacteria, DNA methylation protects the host from foreign DNA through the action of restriction modification systems composed of a restriction endonuclease and a DNA modification methyltransferase (MTase). 1The abbreviations used are: MTase, methyltransferase; AdoMet, S-adenosyl-l-methionine; AdoHcy, S-adenosyl-l-homocysteine; M.RsrI, RsrI DNA methyltransferase; PDB, Protein Data Bank; r.m.s.d., root-mean-squared deviations; 5′-MTA, 5′-methylthioadenosine; TRD, target recognition domain. Both enzymes recognize a specific DNA sequence that is usually palindromic. The endonuclease cleaves the DNA unless the MTase has added a methyl group to a base in the recognition sequence. Restriction modification systems are classified by their subunit composition and cofactor requirements (4Bujnicki J.M. Acta Biochim. Pol. 2001; 48: 969-983Crossref PubMed Scopus (9) Google Scholar). The most studied of these classes, type II restriction modification systems, usually have a dimeric endonuclease and a monomeric MTase that requires only the methyl-donating cofactor S-adenosyl-l-methionine (AdoMet) for activity. Type II DNA MTases share a conserved catalytic core structure (5Roberts R.J. Belfort M. Bestor T. Bhagwat A.S. Bickle T.A. Bitinaite J. Blumenthal R.M. Degtyarev S.K. Dryden D.T.F. Dybvig K. Firman K. Gromova E.S. Gumport R.I. Halford S.E. Hattman S. Heitman J. Hornby D.P. Janulaitis A. Jeltsch A. Josephsen J. Kiss A. Klaenhammer T.R. Kobayashi I. Kong H. Kruger D.H. Lacks S. Marinus M.G. Miyahara M. Morgan R.D. Murray N.E. Nagaraja V. Piekarowicz A. Pingoud A. Raleigh E. Rao D.N. Reich N. Repin V.E. Selker E.U. Shaw P.C. Stein D.C. Stoddard B.L. Szybalski W. Trautner T.A. Van Etten J.L. Vitor J.M. Wilson G.G. Xu S.Y. Nucleic Acids Res. 2003; 31: 1805-1812Crossref PubMed Scopus (566) Google Scholar), composed of a seven-β-stranded sheet flanked by three α-helices on each side, that contains the ligand-binding pockets and active site (6Gong W. O'Gara M. Blumenthal R.M. Cheng X. Nucleic Acids Res. 1997; 25: 2702-2715Crossref PubMed Scopus (170) Google Scholar, 7Malone T. Blumenthal R.M. Cheng X. J. Mol. Biol. 1995; 253: 618-632Crossref PubMed Scopus (439) Google Scholar). The amino acid sequences of the MTases are not as well conserved as their structures, but they share nine conserved sequence motifs (7Malone T. Blumenthal R.M. Cheng X. J. Mol. Biol. 1995; 253: 618-632Crossref PubMed Scopus (439) Google Scholar). On the basis of these motifs, the MTases are subdivided into C5 MTases, which methylate cytosine at C5, and α, β, and γ subclasses of amino MTases, which methylate either adenine at the N6 position (N6A) or cytosine at the N4 position (N4C) (7Malone T. Blumenthal R.M. Cheng X. J. Mol. Biol. 1995; 253: 618-632Crossref PubMed Scopus (439) Google Scholar). Of the conserved motifs, the catalytic motif IV, which contains a conserved PC for the C5 MTases (8Wu J.C. Santi D.V. J. Biol. Chem. 1987; 262: 4778-4786Abstract Full Text PDF PubMed Google Scholar) and a conserved (D/N/S)PP(Y/F) for the amino MTases (7Malone T. Blumenthal R.M. Cheng X. J. Mol. Biol. 1995; 253: 618-632Crossref PubMed Scopus (439) Google Scholar), is implicated in catalysis (9Schubert H.L. Blumenthal R.M. Cheng X. Trends Biochem. Sci. 2003; 28: 329-335Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar). RsrI MTase (M.RsrI) is a β-class N6A enzyme that recognizes the palindromic duplex DNA sequence GAATTC and methylates the internal adenine on each strand (10Kaszubska W. Aiken C. O'Connor C.D. Gumport R.I. Nucleic Acids Res. 1989; 17: 10403-10425Crossref PubMed Scopus (39) Google Scholar, 11Kaszubska W. Webb H.K. Gumport R.I. Gene (Amst.). 1992; 118: 5-11Crossref PubMed Scopus (23) Google Scholar). It is an isoenzyme of EcoRI MTase, a γ-class MTase, with which it shares very little sequence homology (16% identity (10Kaszubska W. Aiken C. O'Connor C.D. Gumport R.I. Nucleic Acids Res. 1989; 17: 10403-10425Crossref PubMed Scopus (39) Google Scholar)). M.RsrI is most homologous in structure and sequence identity (28%) to the N4C MTase PvuII (6Gong W. O'Gara M. Blumenthal R.M. Cheng X. Nucleic Acids Res. 1997; 25: 2702-2715Crossref PubMed Scopus (170) Google Scholar). Previous work partially characterized kinetic and binding properties of M.RsrI with AdoMet, as well as the product of the methylation reaction S-adenosyl-l-homocysteine (AdoHcy) and the specific inhibitor sinefungin (12Szegedi S.S. Reich N.O. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar). The structure of M.RsrI a product of AdoMet, J.L. 25: PubMed Scopus Google Scholar) in the ligand-binding site R.D. S. A. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar). the structures of RsrI MTase bound to the ligands AdoMet, AdoHcy, or The structures the and in ligand binding and explain observed the structure of the L72P mutant of M.RsrI S.S. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar), which is the first DNA MTase structure to determined without bound ligand. structure a unique to the changes of DNA MTases that ligand the L72P in a genetic S.S. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar). The used in the structure by R.D. S. A. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar, S.S. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar). with the used to the L72P into the The an which used to for the The of the and the of changes were by DNA and M.RsrI and were from R.D. S. A. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar) or the mutant and as (12Szegedi S.S. Reich N.O. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar, R.D. S. A. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar). AdoMet from AdoHcy from and sinefungin a from three were used without and were determined an of at M.RsrI by the of AdoMet, AdoHcy, or sinefungin at and in a of the as the and had of R.D. S. A. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar). of the were for in the well to for were the but without the of ligand, and in of Data and of each used to and and at The L72P and for the at a of were on and at In by the through in and were at The of the of the are Data were the W. 1997; PubMed Scopus Google Scholar), and W. 1997; PubMed Scopus Google Scholar), or of and and are for and of the not the only used for and are for and of the ligand from and 1997; PubMed Scopus Google Scholar) are for and of the not the only used for in a of the M.RsrI with the of of the active site were for the of the ligands. The ligand and were T.A. M. Acta A. PubMed Scopus Google Scholar). The were the in and J. M. R.J. T. Acta Biol. PubMed Scopus Google Scholar). of and the of the J.M. J. Google Scholar) and J. M. R.J. T. Acta Biol. PubMed Scopus Google Scholar) were used to the the of ligand in the L72P ligand in the and the determined to were used to the the wild-type of MTase structures were from the Protein Data M.RsrI, R.D. S. A. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google M. N. E. W. J. Mol. Biol. 1997; PubMed Scopus Google M. N. E. W. J. Mol. Biol. 1997; PubMed Scopus Google M. N. E. W. J. Mol. Biol. 1997; PubMed Scopus Google (6Gong W. O'Gara M. Blumenthal R.M. Cheng X. Nucleic Acids Res. 1997; 25: 2702-2715Crossref PubMed Scopus (170) Google M. X. R.J. Cheng X. J. Mol. Biol. PubMed Scopus Google S. S.S. Lacks Full Text Full Text PDF PubMed Scopus Google Scholar). The root-mean-squared structures were determined the N. 1997; PubMed Scopus Google Scholar). The T.A. M. Acta A. PubMed Scopus Google Scholar) and The were determined by The were J. Google Scholar), N. 1997; PubMed Scopus Google Scholar), 1997; PubMed Scopus Google Scholar), and of the structures were in the Protein Data the the of RsrI MTase, M.RsrI with the substrate AdoMet, the product AdoHcy, or the inhibitor The and the structures were by the determined structure R.D. S. A. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar). for the amino and the amino the amino and for the and amino and for the AdoHcy or sinefungin The for each ligand well the structures are to each other and to the structure as by the of the and distinct were observed the bound ligand structures with either each other or to the structure of M.RsrI bound to the AdoMet product R.D. S. A. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google and in a in to the three structures the of the or of the bound ligand and In the AdoMet the had a of which the toward the of the from the adenine conformation the activated methyl group that it toward the catalytic DPPY and the active site of the enzyme The were and for AdoHcy and sinefungin, These the of the ligand for AdoHcy or for in the as the conformation of the and with the catalytic DPPY an that is not observed in the AdoMet complex. In the the group of sinefungin, to the activated methyl group in AdoMet, toward the of the and of the the methyl group of AdoMet The binding pocket contains and a chloride ion at the position by a of the chloride a to the of and to the of and near the target recognition of the from observed of the chloride by that site The chloride ion in the ligand-binding pocket not with but it is that to site by the bound of DNA are AdoHcy, sinefungin, and The to the of the moiety are with for In to in of the AdoHcy and sinefungin the conformations from of AdoMet. Both AdoHcy and sinefungin had with for AdoMet and of the the and and the only in the adenine and were the and which are to the these in conformations to by the in the The of a or (AdoHcy), to the (AdoMet) or by may to the conformations these in the M.RsrI structures in sinefungin to the of the activated methyl group in The adenine of AdoMet and sinefungin that of AdoHcy from the to the other The binding of the moiety of the three ligands by M.RsrI with the ligands bound to as in the ligand moiety are from M. N. E. W. J. Mol. Biol. 1997; PubMed Scopus Google of AdoMet, AdoHcy, and sinefungin with in a of the ligands bound to M.RsrI with bound to other DNA MTases a of and but the of AdoMet are to the of the other ligands AdoMet, with the of the a of in of the structures, which may to the common bent conformation and the of the methyl With the of the AdoMet structures have a bent the AdoHcy and sinefungin structures an In the of the bound AdoHcy a bent conformation to that of AdoMet. AdoHcy in the for the PvuII structure only of methyl group as the were in the of AdoMet and the structure as AdoMet were bound (6Gong W. O'Gara M. Blumenthal R.M. Cheng X. Nucleic Acids Res. 1997; 25: 2702-2715Crossref PubMed Scopus (170) Google Scholar). of ligand binding the of AdoMet as the ligand bound to as AdoMet is the only ligand that with a bent conformation in of the other MTase in methyltransferase for with in the and each with in the and each with in the and each with in the and each with in the and each with in the and each for with in the and each in a In to in ligand M.RsrI amino acid the ligands position and structure and the and that the most is in the AdoHcy and sinefungin structures, had into the position by the amino group of the AdoMet The of in the AdoMet structure is most to by the group of the AdoMet M.RsrI in the AdoHcy for from the and structures at and by and Both of these amino the adenine of the ligand, and their in position the of the adenine in the AdoHcy of the AdoMet, AdoHcy, and M.RsrI structures to the M.RsrI which has in the active reveals changes in the enzyme structure the ligand-binding site R.D. S. A. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar). The the AdoMet structure and structure only are in II three structures from the structure in The the structures in the to the ligand-binding site the loop that the of the bound ligand and occupies a position in the structure to the of a on that ligand. The other the structures were in of and with structures of DNA MTases bound ligand. it to that a from a S.S. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar), not and with bound ligand. mutant DNA but the activity of the enzyme is by at S.S. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar). The L72P mutant of RsrI MTase and the structure by The structure amino and and the for the of the structure an of for the with the complex. The ligand-binding site ligand and with added ligand a of the the of in the ligand-binding site were as In the the L72P the a in the which to a of the on of the and a of the of an on the other these changes were not the of the in to have to changes in the The by the to a in the the catalytic DPPY from the ligand-binding site and a of in the conserved The by the L72P the the L72P to from the ligand-binding site and toward the of the The in the in which of the of the ligand-binding from its position in the AdoMet structure by the of the ligand-binding The loop and to the L72P loop on the active site were from the ligand-binding the and the movement of to the of including from the has from its position in the AdoMet the most the and the L72P structures the ligand-binding loop on the of the binding site from the L72P loop from the ligand-binding site by and with the in an in the of the ligand-binding site from and in the AdoMet structure to in the L72P structure The the in the AdoMet structure and that the of ligand responsible for its the in these a motif that is conserved many of the β-class of amino MTases, but not (7Malone T. Blumenthal R.M. Cheng X. J. Mol. Biol. 1995; 253: 618-632Crossref PubMed Scopus (439) Google Scholar). and a of β-class MTases a motif at the position (7Malone T. Blumenthal R.M. Cheng X. J. Mol. Biol. 1995; 253: 618-632Crossref PubMed Scopus (439) Google Scholar). of the in M.RsrI with in and other MTases may the and of the ligand-binding pocket, and chloride ion not bind in the other structure of M.RsrI little binding of AdoMet, AdoHcy, and from changes in the the only changes were observed in the ligand-binding loop which to the of AdoMet. The of these changes not the observed ligand-binding are in the ligand In the of the ligand for in the in larger of the of the bound ligands in the M.RsrI structures with of structures well conserved conformations DNA MTases by the were observed for the structures, which is not the conserved of the ligand binding and active (7Malone T. Blumenthal R.M. Cheng X. J. Mol. Biol. 1995; 253: 618-632Crossref PubMed Scopus (439) Google Scholar). the AdoMet structures by AdoMet in the DNA MTases, AdoMet in and MTases (9Schubert H.L. Blumenthal R.M. Cheng X. Trends Biochem. Sci. 2003; 28: 329-335Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar). The adenine and of the ligands in the M.RsrI structures bound in is in to the only other MTase with of the adenine and are to by to the enzymes is to the of of the ligand-binding site M. N. E. W. J. Mol. Biol. 1997; PubMed Scopus Google Scholar). The of the to may the in the ligand-binding for the of ligand binding by MTases, including M.RsrI, to from the in the position of the on the three ligands. The of AdoMet and the group of sinefungin a AdoHcy is at the position In the AdoMet and sinefungin structures, which is conserved in many β-class MTases (7Malone T. Blumenthal R.M. Cheng X. J. Mol. Biol. 1995; 253: 618-632Crossref PubMed Scopus (439) Google Scholar), a to the In for to the of AdoMet a bent the in position of the on the in AdoMet with the position of the in sinefungin the in sinefungin to the by the to AdoHcy a its is not to into the pocket by the of a to the of to The conformations of the ligand are not in the ligand (12Szegedi S.S. Reich N.O. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar). The for AdoMet and AdoHcy were very AdoHcy three to the with and with a of which is to that of AdoMet (12Szegedi S.S. Reich N.O. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar). In sinefungin AdoMet are and has which to its affinity of by the binding of and of sinefungin to the and are may explain the in observed sinefungin and AdoMet (12Szegedi S.S. Reich N.O. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar). The movement of the adenine and and in the structure may to the in for AdoHcy to AdoMet. The of ligand binding by the the and structures an the of the in the as observed for the inhibitor sinefungin and the product AdoHcy, base studies to base DNA at the target in the of sinefungin, show an in with the base of the and into the active site (12Szegedi S.S. Reich N.O. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar). Two are with The position observed for sinefungin and AdoHcy in the structure not with base that the ligand to its observed the active site contains the target the ligand the as AdoMet DNA to the complex. In the ligand to bind to DNA binding on the (12Szegedi S.S. Reich N.O. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar). the is the for the conformation in the structure the conformation were in an of the bound ligands. observed an ligand conformation in either the sinefungin or AdoHcy structures, without a structure of the enzyme bound to DNA and sinefungin or of and structures a chloride in the position by the group of the in the structure with bound AdoMet the of chloride with AdoMet DNA M.RsrI activity is to and the enzyme is (10Kaszubska W. Aiken C. O'Connor C.D. Gumport R.I. Nucleic Acids Res. 1989; 17: 10403-10425Crossref PubMed Scopus (39) Google Scholar). The EcoRI MTase not show inhibition by at but not M.RsrI in a methylation not that the effect is and not to the of a chloride in the active site pocket of AdoHcy, and sinefungin a for enzyme inhibition by kinetic studies to the of the inhibition observed it to the chloride that observed in the The chloride ion in the structures to a in the of the target recognition The from the of the protein, and the chloride is of a and in the of the and in the chloride may partially responsible for enzyme inhibition as it the of the DNA with the of the in S.S. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar). is in a in the TRD, the of which to a of DNA binding in a S.S. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar). The bent conformation of the of AdoMet the of the enzyme. DNA amino MTases the methyl group from AdoMet to the target base with of of the methyl A. Santi D.V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). requires the methyl group in the enzyme active site near the target the reaction by an A. Santi D.V. J. Biol. Chem. Full Text PDF PubMed Google Scholar), a of the the methyl and the of is observed for the bound AdoMet. In the structure implicated the conserved in motif in the amino group of the target base as to the methyl group K. M. E. Biol. 2001; PubMed Scopus Google Scholar). the and the to the amino group of the adenine base of the target sequence. In the structure of RsrI MTase with AdoMet, the amino group of the and the of interactions with of the of the catalytic These interactions position the catalytic in the to and help the amino group on the target The may as the for the from the adenine methyl in L72P structure a first into the changes that ligand binding and a for the affinity for specific DNA the ligand is bound to the enzyme. is ligand binding by M.RsrI, and other MTases, DNA binding affinity (12Szegedi S.S. Reich N.O. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar, N.O. N. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Hattman S. Nucleic Acids Res. 1997; 25: Scopus Google Scholar). The M.RsrI without added ligand, but observed bound in the active to with the enzyme R.D. S. A. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar). that to other in DNA binding in a an amino acid the interactions of the adenine and with the enzyme to to most of the ligand-binding loop into position The L72P structure a bound ligand either the ligand not or the enzyme not with bound ligand. The is most as have been to of the L72P mutant in the of added AdoMet. The M.RsrI ligand-binding loop is on the of the ligand-binding site from the L72P and other in the which it to the of the ligand-binding of the movement of loop is that the ligand is responsible for the loop in the position observed in the structure of the wild-type protein, the of the loop it for the in the The in the structure is important in DNA and ligand binding K. M. E. Biol. 2001; PubMed Scopus Google Scholar). the changes that ligand loop in M.RsrI is in DNA as In mechanisms AdoMet which is the for N.O. N. PubMed Scopus Google Scholar) and is the for M.RsrI (12Szegedi S.S. Reich N.O. Gumport R.I. Nucleic Acids Res. 2000; 28: PubMed Google Scholar), the binding of ligand the ligand-binding loop and other into a position that DNA In which may have a with a of binding E. S. E. S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) or a DNA first J. Reich N.O. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), to have binding for DNA and AdoMet that of the ligand is not for binding ligand binding affinity for specific DNA binding in but has been as an of an in the enzyme J. Reich N.O. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The structure of the L72P mutant that changes by subtle of the structures on either of the ligand The most amino acid responsible for the binding of ligand are the DPPY motif and the which from the they in the AdoMet movement the ligand-binding most to ligand affinity and the of the ligand affinity with the movement of the DPPY motif explain the catalytic activity of the L72P for the activity of the L72P mutant from the that in the structures of or the the loop to the loop the M.RsrI L72P is in DNA binding the base K. M. E. Biol. 2001; PubMed Scopus Google Scholar, S. S. R.J. Cheng X. Full Text PDF PubMed Scopus Google Scholar). The L72P the loop or conformation in a as to either the base either in a conformation or the of the DNA and enzyme kinetic studies to the of these to the activity structure of the L72P mutant and of the wild-type enzyme with the substrate, AdoMet, the product, AdoHcy, and the inhibitor sinefungin into ligand binding by with other MTases a in ligand-binding The of a chloride in the active site pocket of AdoHcy, and sinefungin a for the inhibition of M.RsrI by the ligand in the L72P structure a first into the changes that ligand the of in for the AdoHcy A. and at the at for their help with for at the and X. Cheng for (9Schubert H.L. Blumenthal R.M. Cheng X. Trends Biochem. Sci. 2003; 28: 329-335Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar) to The of core is by the of and the