Abstract
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.
Highlights
Kre2p/Mnt1p is a Golgi ␣1,2-mannosyltransferase involved in the biosynthesis of Saccharomyces cerevisiae cell wall glycoproteins
The P. pastoris expression kit was obtained from Invitrogen; the QuikChangeTM site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA); GDP-[6-3H]mannose (40 Ci/mmol) was from American Radiolabeled Chemicals Inc
MO); GDP-mannose and methyl-␣-mannoside were from Sigma Chemical Co; Macro-Prep ceramic hydroxyapatite type I 20 m and AG 1-X8 resin were from Bio-Rad (Misssissauga, Ontario, Canada); oligonucleotides were synthesized by Biocorp Inc. (Montreal, Quebec, Canada)
Summary
The P. pastoris expression kit was obtained from Invitrogen; the QuikChangeTM site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA); GDP-[6-3H]mannose (40 Ci/mmol) was from American Radiolabeled Chemicals Inc. (St. Louis, MO); GDP-mannose and methyl-␣-mannoside were from Sigma Chemical Co; Macro-Prep ceramic hydroxyapatite type I 20 m and AG 1-X8 resin were from Bio-Rad (Misssissauga, Ontario, Canada); oligonucleotides were synthesized by Biocorp Inc. All other chemicals were of reagent grade
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