Mechanism of Mg2+-Accompanied Product Release in Sugar Nucleotidyltransferases

• Published in: Structure (London) 2018-03, Vol.26 (3), p.459-466.e3
• Main Authors: Vithani, Neha, Ankush Jagtap, Pravin Kumar, Verma, Sunil Kumar, Tripathi, Ravi, Awasthi, Shalini, Nair, Nisanth N., Prakash, Balaji
• Format: Article
• Language: English
• Subjects: enhanced sampling molecular dynamics simulations; free energy computations; Mg2+ ion; nucleotidyl transfer; product release; structural snapshots; uridyltransfer reaction; X-ray crystallography
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2018.02.002

The nucleotidyl transfer reaction, catalyzed by sugar nucleotidyltransferases (SNTs), is assisted by two active site Mg2+ ions. While studying this reaction using X-ray crystallography, we captured snapshots of the pyrophosphate (product) as it exits along a pocket. Surprisingly, one of the active site Mg2+ ions remains coordinated to the exiting pyrophosphate. This hints at the participation of Mg2+ in the process of product release, besides its role in catalyzing nucleotidyl transfer. These observations are further supported by enhanced sampling molecular dynamics simulations. Free energy computations suggest that the product release is likely to be rate limiting in SNTs, and the origin of the high free energy barrier for product release could be traced back to the “slow” conformational change of an Arg residue at the exit end of the pocket. These results establish a dual role for Mg2+, and propose a general mechanism of product release during the nucleotidyl transfer by SNTs. [Display omitted] •Sugar nucleotidyltransferases (SNTs) utilize two Mg2+ ions for catalysis•Structural snapshots depict the second Mg2+ participating in product release•Computational studies identify the mechanism for the exit of product-Mg2+ complex•Change in side-chain conformation of an Arg residue is rate limiting Sugar nucleotidyltransferases use two magnesium ions for catalysis. Vithani, Jagtap, Verma et al. show that the second magnesium ion is important not only for catalysis (converting substrates to products) but also for product release. Using experimental and computational studies, the authors show that product release is critical in these enzymes.