Abstract

ABSTRACTThe nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74‐compound library of known Nudix enzyme substrates. We found substrates for four enzymes with k cat/K m values >10,000 M−1 s−1: Q92EH0_LISIN of Listeria innocua serovar 6a against ADP‐ribose, Q5LBB1_BACFN of Bacillus fragilis against 5‐Me‐CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8‐oxo‐dATP and 3'‐dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty‐two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported k cat/K m values exhibited against these canonical substrates are well under 105 M−1 s−1. By contrast, several Nudix enzymes show much larger k cat/K m values (in the range of 105 to >107 M−1 s−1) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810–1822. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Highlights

  • The Nudix protein superfamily is vast and diverse.[1,2]It comprises about 50,000 members in the Nudix clan (Pfam ID: CL0261) of the Pfam database,[3,4] and all members share a characteristic 130 amino acid beta-grasp domain architecture[5] classified as the Nudix fold (SCOPe v2.03 SUNID 55810, SCCSID d.113;6,7)

  • Many Nudix proteins are pyrophosphohydrolases. They catalyze the hydrolysis of nucleoside diphosphates linked to some other moiety, X.1. These Nudix hydrolases are typically characterized by a sequence of 23 amino acids [Gx5Ex7REUxEExGU], where U can be Ile, Leu, or Val, and x represents any amino acid

  • The mean kcat/Km values of each group is represented by a white bar

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Summary

Introduction

The Nudix protein superfamily is vast and diverse.[1,2]. It comprises about 50,000 members in the Nudix clan (Pfam ID: CL0261) of the Pfam database (version 27.0),[3,4] and all members share a characteristic 130 amino acid beta-grasp domain architecture[5] classified as the Nudix fold (SCOPe v2.03 SUNID 55810, SCCSID d.113;6,7). They catalyze the hydrolysis of nucleoside diphosphates linked to some other moiety, X.1. These Nudix hydrolases are typically characterized by a sequence of 23 amino acids [Gx5Ex7REUxEExGU], where U can be Ile, Leu, or Val, and x represents any amino acid.

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