Evidence for a Two-Metal-Ion Mechanism in the Cytidyltransferase KdsB, an Enzyme Involved in Lipopolysaccharide Biosynthesis 英文参考文献.docVIP

EvidenceforaTwo-Metal-IonMechanisminthe CytidyltransferaseKdsB,anEnzymeInvolvedin LipopolysaccharideBiosynthesis HelgoSchmidt1.¤a,JeroenR.Mesters1.,JingWu2¤b,RonaldW.Woodard2,RolfHilgenfeld1,3,4*, UweMamat5* 1InstituteofBiochemistry,CenterforStructuralandCellBiologyinMedicine,UniversityofLu¨beck,Lu¨beck,Germany,2DepartmentofMedicinalChemistry,Collegeof Pharmacy,University of Michigan, Ann Arbor, Michigan, United States ofAmerica, 3Laboratory for Structural Biology ofInfectionandInflammation, DESY, Hamburg, Germany,4ShanghaiInstituteofMateriaMedica,ChineseAcademyofSciences,Shanghai,China,5DivisionofStructuralBiochemistry,ResearchCenterBorstel,Leibniz- CenterforMedicineandBiosciences,Borstel,Germany Abstract Lipopolysaccharide (LPS) is located on the surface of Gram-negative bacteria and is responsible for maintaining outer membranestability,whichisaprerequisiteforcellsurvival.Furthermore,itrepresentsanimportantbarrieragainsthostile environmentalfactorssuchasantimicrobialpeptidesandthecomplementcascadeduringGram-negativeinfections.The sugar3-deoxy-D-manno-oct-2-ulosonicacid(Kdo)isanintegralpartofLPSandplaysakeyroleinLPSfunctionality.Priorto itsincorporationintotheLPSmolecule,KdohastobeactivatedbytheCMP-Kdosynthetase(CKS).Basedonthepresenceof asingleMg2+ ionintheactivesite,detailedmodelsofthereactionmechanismofCKShavebeendevelopedpreviously. 2+ Recently,atwo-metal-ionhypothesissuggestedtheinvolvementoftwoMg ionsinKdoactivation.Tofurtherinvestigate the mechanistic aspects of Kdo activation, we kinetically characterized the CKS from the hyperthermophilic organism Aquifex aeolicus. In addition, we determined the crystal structure of this enzyme at a resolution of 2.10 A? and provide evidencethattwoMg ionsarepartoftheactivesiteoftheenzyme. 2+ Citation: Schmidt H, Mesters JR, Wu J, Woodard RW, Hilgenfeld R, et al. (2011) Evidence for a Two-Metal-Ion Mechanism in the Cytidyltransferase KdsB, an EnzymeInvolvedinLipopolysaccharideBiosynthesis.PLoSONE6(8):e23231.doi:1