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Yeast Mnn9 is both a priming glycosyltransferase and an allosteric activator of mannan biosynthesis.

ABSTRACT: The fungal cell possesses an essential carbohydrate cell wall. The outer layer, mannan, is formed by mannoproteins carrying highly mannosylated O- and N-linked glycans. Yeast mannan biosynthesis is initiated by a Golgi-located complex (M-Pol I) of two GT-62 mannosyltransferases, Mnn9p and Van1p, that are conserved in fungal pathogens. Saccharomyces cerevisiae and Candida albicans mnn9 knockouts show an aberrant cell wall and increased antibiotic sensitivity, suggesting the enzyme is a potential drug target. Here, we present the structure of ScMnn9 in complex with GDP and Mn(2+), defining the fold and catalytic machinery of the GT-62 family. Compared with distantly related GT-78/GT-15 enzymes, ScMnn9 carries an unusual extension. Using a novel enzyme assay and site-directed mutagenesis, we identify conserved amino acids essential for ScMnn9 'priming' ?-1,6-mannosyltransferase activity. Strikingly, both the presence of the ScMnn9 protein and its product, but not ScMnn9 catalytic activity, are required to activate subsequent ScVan1 processive ?-1,6-mannosyltransferase activity in the M-Pol I complex. These results reveal the molecular basis of mannan synthesis and will aid development of inhibitors targeting this process.

SUBMITTER: Striebeck A

PROVIDER: S-EPMC3787745 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Yeast Mnn9 is both a priming glycosyltransferase and an allosteric activator of mannan biosynthesis.

Striebeck Alexander A Robinson David A DA Schüttelkopf Alexander W AW van Aalten Daan M F DM

Open biology 20130911 9

The fungal cell possesses an essential carbohydrate cell wall. The outer layer, mannan, is formed by mannoproteins carrying highly mannosylated O- and N-linked glycans. Yeast mannan biosynthesis is initiated by a Golgi-located complex (M-Pol I) of two GT-62 mannosyltransferases, Mnn9p and Van1p, that are conserved in fungal pathogens. Saccharomyces cerevisiae and Candida albicans mnn9 knockouts show an aberrant cell wall and increased antibiotic sensitivity, suggesting the enzyme is a potential ...[more]

PMID: 24026536

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Project description:A variant form of yeast pyruvate kinase (EC 2.7.1.40) with Ser-384 mutated to proline has been engineered in order to study the allosteric properties of this enzyme. Both the mutant and wild-type enzymes were overexpressed in a strain of yeast in which the genomic copy of the pyruvate kinase gene had been disrupted by an insertion of the Ura3 gene. Both enzymes were purified to homogeneity and their kinetic properties characterized. The wild-type enzyme displays sigmoid kinetics with respect to phosphoenolpyruvate (PEP) concentration, and is activated by the allosteric effect fructose 1,6-bisphosphate with concomitant reduction in co-operativity. In contrast, the mutant was found to be dependent on the presence of the effector for catalytic activity and was inactive in its absence. The fully activated mutant enzyme had a kcat. 1.6 times greater than that of the wild-type enzyme. The mutation introduced into the enzyme is in an intersubunit contact which is known to be critical for the allosteric properties of the enzyme, and is far removed from the active site. The major effect of the mutation seems to be to stabilize the low-affinity T state of the apoenzyme, although kcat. is also affected. The S0.5 for PEP and S0.5 for ADP of the wild-type enzyme were 0.22 +/- 0.004 and 0.15 +/- 0.01 mM respectively (means +/- S.E.M.). In the activated mutant enzyme, these kinetic parameters increased to 0.67 +/- 0.03 and 0.43 +/- 0.03 mM respectively. The cooperativity between ADP-binding sites was altered in the mutant enzyme, with the Hill coefficient (h) for ADP increasing to 1.65 +/- 0.07 in the presence of the effector, compared with a value of 0.01 +/- 0.07 for the wild-type enzyme under the same conditions. CD spectroscopy revealed the secondary structure of the mutant enzyme to be little different from that of the wild-type enzyme, indicating that the two enzymes have similar secondary structures in solution. Precise tertiary and quaternary structures such as intersubunit and interdomain interactions may be modified. An improved purification procedure has been devised that allows large quantities of enzyme to be rapidly prepared.

Dataset Information

Yeast Mnn9 is both a priming glycosyltransferase and an allosteric activator of mannan biosynthesis.

Publications

Yeast Mnn9 is both a priming glycosyltransferase and an allosteric activator of mannan biosynthesis.

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