Structure and mechanism to function: allosteric activation of phosphomannomutase 1 and substrate selectivity in Hotdog Fold thioesterases
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Two superfamilies were used to explore the structure/function relationship as it pertains to enzyme specificity. The structures and mechanisms of phosphatases belonging to the Haloalkanoate Dehalogenase Superfamily (HADSF) and thioesterases of the Hotdog Fold Superfamily (HDFSF) were determined using X-ray crystallography and other biophysical tools in combination with steady-state kinetics and site-directed mutagenesis. Together, the specific structural components of enzymes crucial for substrate recognition, substrate promiscuity, and catalysis were uncovered. In the HADSF, the phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate and mannose 1-phosphate, an essential step in the protein glycosylation pathway. In humans, two isoforms PMM1 and PMM2 catalyze this reaction. Deficiency in PMM2 activity is the major cause of congenital disorders of glycosylation (CDG-1a). However, PMM1 activity is not sufficient to replace PMM2 in protein glycosylation. Instead, PMM1 functions as a glucose-1,6-bisphosphate phosphatase in the presence of IMP and enables the temporary rescue of glycolysis during brain ischemia. Herein, the structure of IMP bound to PMM1 in combination with kinetics revealed a mechanism for the differential substrate preference and the mechanistic switch from a mutase to phosphatase activity. In the HDFSF, the majority of which function as thioesterases of aliphatic or aromatic compounds bound to coenzyme A or acyl carrier protein (ACP), the structural determinants for substrate preference were identified in PA1618, an enzyme with high substrate promiscuity. The structural determinants of the specific thiosterases MA0038 and BVU1957 were also identified. The variation in substrate range observed among these enzymes, from specific to promiscuous, led to the design of a comprehensive thioester screen to identify HDFSF thioesterase substrates. The profiles of substrate specificities from 42 previously uncharacterized thioesterases were determined allowing comparison of the sequence/substrate relationships across a representative selection of thioesterases. Examples drawn from both HADSF and HDFSF enzymes suggested a model relating substrate promiscuity and specificity to regions of protein flexibility. The motion of a domain within a multidomain protein or a flexible loop near the active site was correlated with the occurrence of substrate promiscuity/specificity.