Structure guided insight into function, mechanism, and evolution in a phosphoryl- and phosphoglycosyl- transferase superfamily
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Abstract
The haloalkanoate dehalogenase (HAD) and monotopic phosphoglycosyl transferase (monoPGT) superfamilies were used to study structure/function relationships and protein evolution. Phosphomannomutases (PMM), members of the HAD superfamily, catalyze the transformation of mannose 6-phosphate to mannose 1-phosphate, an essential reaction in eukaryotic N-linked glycosylation. In mammals, there are two isozymes of PMM which share high sequence identity (67% for human isozymes). PMM2 is ubiquitously expressed, and mutations to this gene cause congenital diseases of glycosylation (CDGs), resulting in depleted levels of glycoconjugate products (hypoglycosylation). In contrast, PMM1 is expressed in the brain, lungs, and kidneys, and no measurable phenotype is detected in mouse knockout models. In vitro characterization of PMM isozymes provide a proposed link of isozyme specificity to disease-related phenotypes. Clinically, galactosemia patients exhibit buildup of galactose 1-phosphate (Gal1P) and phenotypes similar to those seen in CDGs including hypoglycosylation. In this work we showed that in vitro, Gal1P inhibits PMM2 at physiological concentrations. However, Gal1P does not inhibit PPM1, even at saturating concentrations.
The X-ray crystal structure of PMM1 in complex with vanadate reveals distinct preference for binding to the distal phosphoryl sub-site. Combined with in vitro inhibition analysis of PMM1 by vanadate, the structures support a “catch and close” mechanism of substrate binding, rather than an induced fit model.
A superfamily approach yielded insight into protein evolution and substrate specificity in the monoPGT superfamily. MonoPGTs catalyze coupling of a soluble nucleotide-activated sugar to a membrane-resident polyprenol phosphate (PrenP), the first membrane-committed step in glycoconjugate biosynthetic pathways. The diversity of the family is revealed by the generation and analysis of a sequence-similarity network (SSN), with fusion of monoPGTs with other pathway members being the most frequent and extensive elaboration. The fusion domains fall into three subclasses: sugar-modifying enzymes, glycosyl transferases, and regulatory domains. The occurrence of protein fusions in the glycoconjugate biosynthetic pathways provides support for protein-protein interactions that could increase pathway throughput. Analyses of the re-entrant membrane helix (RMH) motif of monoPGTs via generation of hidden Markov models (HMM) describes sequence patterns which may play a role in the positioning and binding of the lipid substrate.