Identification of Residue Involved in Nucleotidyltransferase Activity of LinA from Staphylococci by Site-directed Mutagenesis
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摘要
The lincosamide class of antibacterials is widely used for the treatment of a broad spectrum of infections, and one prevalent route of resistance to lincosamides in pathogenic gram-positive cocco is antibiotic modification. Enzymes encoded by lin genes, belonging to nucleotidyltransferase superfamily, catalyze adenylylation to inactivate lincosamides. LinA can adenylylate lincosamides at either 3?-or 4?-OH of the methylthiolincosamide sugar. The crystal structure of LinA/lincomycin has confirmed its active site. However, the residue interacting with nucleotidyl donors remains elusive. Here, we modeled the complex structure of LinA/lincomycin/Mg~(2+)/AMPCPP to reveal a putative pocket for nucleotidyl donors and suggested the residue R45 in this pocket involved in the recognition of donor substrates NTP and catalysis. ITC and enzyme activity assays show that the mutation of residue R45 impairs LinA nucleotidyltransferase activity in vitro. This work provides insights into the molecular mechanism of the nucleotide binding and transferring activity of antibiotic NTases.
The lincosamide class of antibacterials is widely used for the treatment of a broad spectrum of infections, and one prevalent route of resistance to lincosamides in pathogenic gram-positive cocco is antibiotic modification. Enzymes encoded by lin genes, belonging to nucleotidyltransferase superfamily, catalyze adenylylation to inactivate lincosamides. LinA can adenylylate lincosamides at either 3?-or 4?-OH of the methylthiolincosamide sugar. The crystal structure of LinA/lincomycin has confirmed its active site. However, the residue interacting with nucleotidyl donors remains elusive. Here, we modeled the complex structure of LinA/lincomycin/Mg~(2+)/AMPCPP to reveal a putative pocket for nucleotidyl donors and suggested the residue R45 in this pocket involved in the recognition of donor substrates NTP and catalysis. ITC and enzyme activity assays show that the mutation of residue R45 impairs LinA nucleotidyltransferase activity in vitro. This work provides insights into the molecular mechanism of the nucleotide binding and transferring activity of antibiotic NTases.
The lincosamide class of antibacterials is widely used for the treatment of a broad spectrum of infections, and one prevalent route of resistance to lincosamides in pathogenic gram-positive cocco is antibiotic modification. Enzymes encoded by lin genes, belonging to nucleotidyltransferase superfamily, catalyze adenylylation to inactivate lincosamides. LinA can adenylylate lincosamides at either 3?-or 4?-OH of the methylthiolincosamide sugar. The crystal structure of LinA/lincomycin has confirmed its active site. However, the residue interacting with nucleotidyl donors remains elusive. Here, we modeled the complex structure of LinA/lincomycin/Mg~(2+)/AMPCPP to reveal a putative pocket for nucleotidyl donors and suggested the residue R45 in this pocket involved in the recognition of donor substrates NTP and catalysis. ITC and enzyme activity assays show that the mutation of residue R45 impairs LinA nucleotidyltransferase activity in vitro. This work provides insights into the molecular mechanism of the nucleotide binding and transferring activity of antibiotic NTases.
引文
(1)Morar,M.;Bhullar,K.;Hughes,D.W.;Junop,M.;Wright,G.D.Structure and mechanism of the lincosamide antibiotic adenylyltransferase linB.Structure 2009,17,1649-1659.
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(4)Petinaki,E.;Guerin,Faublee,V.;Pichereau,V.;Villers,C.;Achard,A.;Malbruny,B.;Leclercq,R.Lincomycin resistance gene lnu(D)in streptococcus uberis.Antimicrob.Agents Ch.2008,52,626-630.
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(6)Stogios,P.J.;Evdokimova,E.;Morar,M.;Koteva,K.;Wright,G.D.;Courvalin,P.;Savchenko,A.Structural and functional plasticity of antibiotic resistance nucleotidylyltransferases revealed by molecular characterization of lincosamide nucleotidylyltransferases lnu(A)and lnu(D).J.Mol.Biol.2015,427,2229-2243.
(7)Wright,G.D.Bacterial resistance to antibiotics:Enzymatic degradation and modification.Adv.Drug Deliver.Rev.2005,57,1451-1470.
(8)Sarrou,S.;Liakopoulos,A.;Tsoumani,K.;Sagri,E.;Mathiopoulos,K.D.;Tzouvelekis,L.S.;Miriagou,V.;Petinaki,E.Characterization of a novel lsa(E)-and lnu(B)-carrying structure located in the chromosome of a staphylococcus aureus sequence type 398 strain.Antimicrob.Agents Ch.2016,60,1164-1166.
(9)Singh,K.V.;Weinstock,G.M.;Murray,B.E.An enterococcus faecalis ABC homologue(lsa)is required for the resistance of this species to clindamycin and quinupristin-dalfopristin.Antimicrob.Agents Ch.2002,46,1845-1850.
(10)Long,K.S.;Poehlsgaard,J.;Kehrenberg,C.;Schwarz,S.;Vester,B.The cfr rRNA methyltransferase confers resistance to phenicols,lincosamides,oxazolidinones,pleuromutilins,and streptograrnin a antibiotics.Antimicrob.Agents Ch.2006,50,2500-2505.
(11)Brisson,Noel,A.;Delrieu,P.;Samain,D.;Courvalin,P.Inactivation of lincosaminide antibiotics in staphylococcus.Identification of lincosaminide o-nucleotidyltransferases and comparison of the corresponding resistance genes.J.Biol.Chem.1988,263,15880-15887.
(12)Kuchta,K.;Knizewski,L.;Wyrwicz,L.S.;Rychlewski,L.;Ginalski,K.Comprehensive classification of nucleotidyltransferase fold proteins:Identification of novel families and their representatives in human.Nucleic Acids Res.2009,37,7701-7714.
(13)Emsley,P.;Lohkamp,B.;Scott,W.G.;Cowtan,K.Features and development of coot.Acta Crystallogr.D 2010,66,486-501.
(14)Upson,R.H.;Haugland,R.P.;Malekzadeh,M.N.;Haugland,R.P.A spectrophotometric method to measure enzymatic activity in reactions that generate inorganic pyrophosphate.Anal.Biochem.1996,243,41-45.
(15)Ye,X.R.;Zhao,Y.H.;Wu,X.L.;Su,Y.T.;Wu,Y.K.;She,F.F.Identification of residues involved in nucleotidyltransferase activity of jhp933 from helicobacter pylori by site-directed mutagenesis.Arch.Biol.Sci.2016,68,439-444.
(2)Rezanka,T.;Spizek,J.;Sigler,K.Medicinal use of lincosamides and microbial resistance to them.Anti-Infect.Agents Med.Chem.2007,6,133-144.
(3)Spizek,J.;Rezanka,T.Lincomycin clindamycin and their applications.Appl.Microbiol.Biot.2004,64,455-464.
(4)Petinaki,E.;Guerin,Faublee,V.;Pichereau,V.;Villers,C.;Achard,A.;Malbruny,B.;Leclercq,R.Lincomycin resistance gene lnu(D)in streptococcus uberis.Antimicrob.Agents Ch.2008,52,626-630.
(5)Hoeksema,H.;Magerlein,B.J.;Kagan,F.;Herr,R.R.;Slomp,G.;Birkenmeyer,R.D.;Bannister,B.;Schroeder,W.;Mackellar,F.A.Chemical studies on lincomycin.I.structure of lincomycin.J.Am.Chem.Soc.1964,86,4223-4227.
(6)Stogios,P.J.;Evdokimova,E.;Morar,M.;Koteva,K.;Wright,G.D.;Courvalin,P.;Savchenko,A.Structural and functional plasticity of antibiotic resistance nucleotidylyltransferases revealed by molecular characterization of lincosamide nucleotidylyltransferases lnu(A)and lnu(D).J.Mol.Biol.2015,427,2229-2243.
(7)Wright,G.D.Bacterial resistance to antibiotics:Enzymatic degradation and modification.Adv.Drug Deliver.Rev.2005,57,1451-1470.
(8)Sarrou,S.;Liakopoulos,A.;Tsoumani,K.;Sagri,E.;Mathiopoulos,K.D.;Tzouvelekis,L.S.;Miriagou,V.;Petinaki,E.Characterization of a novel lsa(E)-and lnu(B)-carrying structure located in the chromosome of a staphylococcus aureus sequence type 398 strain.Antimicrob.Agents Ch.2016,60,1164-1166.
(9)Singh,K.V.;Weinstock,G.M.;Murray,B.E.An enterococcus faecalis ABC homologue(lsa)is required for the resistance of this species to clindamycin and quinupristin-dalfopristin.Antimicrob.Agents Ch.2002,46,1845-1850.
(10)Long,K.S.;Poehlsgaard,J.;Kehrenberg,C.;Schwarz,S.;Vester,B.The cfr rRNA methyltransferase confers resistance to phenicols,lincosamides,oxazolidinones,pleuromutilins,and streptograrnin a antibiotics.Antimicrob.Agents Ch.2006,50,2500-2505.
(11)Brisson,Noel,A.;Delrieu,P.;Samain,D.;Courvalin,P.Inactivation of lincosaminide antibiotics in staphylococcus.Identification of lincosaminide o-nucleotidyltransferases and comparison of the corresponding resistance genes.J.Biol.Chem.1988,263,15880-15887.
(12)Kuchta,K.;Knizewski,L.;Wyrwicz,L.S.;Rychlewski,L.;Ginalski,K.Comprehensive classification of nucleotidyltransferase fold proteins:Identification of novel families and their representatives in human.Nucleic Acids Res.2009,37,7701-7714.
(13)Emsley,P.;Lohkamp,B.;Scott,W.G.;Cowtan,K.Features and development of coot.Acta Crystallogr.D 2010,66,486-501.
(14)Upson,R.H.;Haugland,R.P.;Malekzadeh,M.N.;Haugland,R.P.A spectrophotometric method to measure enzymatic activity in reactions that generate inorganic pyrophosphate.Anal.Biochem.1996,243,41-45.
(15)Ye,X.R.;Zhao,Y.H.;Wu,X.L.;Su,Y.T.;Wu,Y.K.;She,F.F.Identification of residues involved in nucleotidyltransferase activity of jhp933 from helicobacter pylori by site-directed mutagenesis.Arch.Biol.Sci.2016,68,439-444.