Structure of Kre2p/Mnt1p: a yeast alpha1,2-mannosyltransferase involved in mannoprotein biosynthesis.
Kre2p/Mnt1p is a Golgi alpha1,2-mannosyltransferase involved in the biosynthesis of Saccharomyces cerevisiae cell wall glycoproteins. The protein belongs to glycosyltransferase family 15, a member of which has been implicated in virulence of Candida albicans. We present the 2.0 A crystal structures of the catalytic domain of Kre2p/Mnt1p and its binary and ternary complexes with GDP/Mn(2+) and GDP/Mn(2+)/acceptor methyl-alpha-mannoside. The protein has a mixed alpha/beta fold similar to the glycosyltransferase-A (GT-A) fold. Although the GDP-mannose donor was used in the crystallization experiments and the GDP moiety is bound tightly to the active site, the mannose is not visible in the electron density. The manganese is coordinated by a modified DXD motif (EPD), with only the first glutamate involved in a direct interaction. The position of the donor mannose was modeled using the binary and ternary complexes of other GT-A enzymes. The C1" of the modeled donor mannose is within hydrogen-bonding distance of both the hydroxyl of Tyr(220) and the O2 of the acceptor mannose. The O2 of the acceptor mannose is also within hydrogen bond distance of the hydroxyl of Tyr(220). The structures, modeling, site-directed mutagenesis, and kinetic analysis suggest two possible catalytic mechanisms. Either a double-displacement mechanism with the hydroxyl of Tyr(220) as the potential nucleophile or alternatively, an S(N)i-like mechanism with Tyr(220) positioning the substrates for catalysis. The importance of Tyr(220) in both mechanisms is highlighted by a 3000-fold reduction in k(cat) in the Y220F mutant.
Lobsanov
Yuri D
YD
Program in Structural Biology and Biochemistry, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
Romero
Pedro A
PA
Sleno
Barry
B
Yu
Bomina
B
Yip
Patrick
P
Herscovics
Annette
A
Howell
P Lynne
PL
D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002473:Cell Wall / Q000378:metabolism; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004583:Electrons; D006023:Glycoproteins / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry; D006860:Hydrogen Bonding; D007700:Kinetics; D008358:Mannose / Q000737:chemistry; D008364:Mannosyltransferases / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D010843:Pichia / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry
D006023:Glycoproteins; D008562:Membrane Glycoproteins; D029701:Saccharomyces cerevisiae Proteins; C025363:mannoproteins; D006153:Guanosine Diphosphate; D014443:Tyrosine; C068701:KRE2 protein, S cerevisiae; D008364:Mannosyltransferases; D008358:Mannose; D010100:Oxygen