Exploiting the rationale behind substrate recognition by promiscuous thermophilic NDP-sugar pyrophosphorylase for expanding glycorandomization: an in silico study.

The fundamental substrates for protein glycosylation are provided by a group of enzymes known as NDP-sugar pyrophosphorylases (NSPases) which utilize nucleotide triphosphate (NTP) and sugar 1-phosphate to catalyze the formation of nucleotide diphospho-sugar (NDP-sugar). The promiscuous nature of NSPases is often exploited during chemoenzymatic glycorandomization in the pursuit of novel therapeutics. However, till date, the number of inherently promiscuous NSPases reported and the rationale behind their promiscuity is meager. In this study, we have identified a set of NSPases from a hyperthermophilic archaeon Pyrococcus horikoshii OT3 to identify probable candidates for glycorandomization. We identified a set of NSPases that include both substrate-specific and substrate-promiscuous NSPases with a visible predominance of the latter group. The rationale behind the promiscuity (or specificity) vividly lies in the repertoire of amino acid residues that assemble the active site for recognition of the substrate moiety. Furthermore, the absence of a function-specific auxiliary domain promotes substrate promiscuity in NSPases. This study, thus, provides a novel set of thermophilic NSPases that can be employed for chemoenzymatic glycorandomization. More importantly, identification of the residues that render substrate promiscuity (or specificity) would assist in sequence-based rational engineering of NSPases for enhanced glycorandomization. Communicated by Ramaswamy H. Sarma.

Structural insights into the catalytic mechanism of 5-methylthioribose 1-phosphate isomerase.

5-Methylthioribose 1-phosphate isomerase (M1Pi) is a crucial enzyme involved in the universally conserved methionine salvage pathway (MSP) where it is known to catalyze the conversion of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P) via a mechanism which remains unspecified till date. Furthermore, although M1Pi has a discrete function, it surprisingly shares high structural similarity with two functionally non-related proteins such as ribose-1,5-bisphosphate isomerase (R15Pi) and the regulatory subunits of eukaryotic translation initiation factor 2B (eIF2B). To identify the distinct structural features that lead to divergent functional obligations of M1Pi as well as to understand the mechanism of enzyme catalysis, the crystal structure of M1Pi from a hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined. A meticulous structural investigation of the dimeric M1Pi revealed the presence of an N-terminal extension and a hydrophobic patch absent in R15Pi and the regulatory α-subunit of eIF2B. Furthermore, unlike R15Pi in which a kink formation is observed in one of the helices, the domain movement of M1Pi is distinguished by a forward shift in a loop covering the active-site pocket. All these structural attributes contribute towards a hydrophobic microenvironment in the vicinity of the active site of the enzyme making it favorable for the reaction mechanism to commence. Thus, a hydrophobic active-site microenvironment in addition to the availability of optimal amino-acid residues surrounding the catalytic residues in M1Pi led us to propose its probable reaction mechanism via a cis-phosphoenolate intermediate formation.