Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family

Abstract

The 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.

Lost in translation: egg transcriptome reveals molecular signature to predict developmental success and novel maternal-effect genes

“…2a-h and Additional file 3, respectively). Literature search showed that most of these genes have known functions: tk2 [36] is a deoxyribonucleoside kinase that is required for mitochondrial DNA synthesis; drd3 [37] is dopamine receptor which is associated with cognitive, emotional, and endocrine functions; cldn23 [38] is an integral membrane protein that maintains cell polarity and signal transductions; nudt13 [39] is hydrolase and its function is also largely unknown; itih2 [40] is a serine protease inhibitor that carries hyaluronan in plasma; flvcr1 [41] is a heme transporter that has a critical role in erythropoiesis by protecting developing erythroid cells from heme toxicity; pomt1 [42] is an O-mannosyltransferase that is a component of many polysaccharides and glycoproteins; prkcq [43] is a serine- and threonine-specific protein kinase that is important for T-cell activation; and otulina [44] is a predicted deubiquitinase also called fam105ba . qPCR confirmed the differential expression of these genes in bad quality eggs relative to good quality eggs (Fig.…”

The evolution of function within the Nudix homology clan

“…Within this category, k cat / K m values, when available, usually provided the most informative conclusion, and thus were assigned with the highest confidence scores. High k cat / K m values (e.g., >10 6 M −1 s −1 ) serve as a sufficient condition to indicate likely physiological substrates, while observation of low k cat / K m values for Nudix hydrolases often means the investigated chemical is not likely the physiological substrate for the enzyme . Hence, we assigned scores of 0.99, 0.85, 0.5, 0.2, and 0.1, corresponding to k cat / K m values 10 7 , 10 6 , 10 5 , 10 4 , 10 3 M −1 s −1 , respectively.…”

Variant Bacterial Riboswitches Associated with Nucleotide Hydrolase Genes Sense Nucleoside Diphosphates

“…Similar NUDIX hydrolase family proteins act on the di-and tri-phosphate versions of other types of damaged DNA and RNA nucleosides, including 8-oxoadenosine, 2-hydroxyadenosine, and 5-hydroxycytosine. 30,32 NUDIX hydrolase family proteins also act on many other nucleotide-containing compounds, such as the enzyme cofactors NADH and TPP, the signaling molecule Ap4A, and the adenylated sugar ADP-glucose. 30,32,33 Some NUDIX hydrolases display broad substrate specificity and act on a range of nucleoside di- and triphosphates.…”

“…30,32 NUDIX hydrolase family proteins also act on many other nucleotide-containing compounds, such as the enzyme cofactors NADH and TPP, the signaling molecule Ap4A, and the adenylated sugar ADP-glucose. 30,32,33 Some NUDIX hydrolases display broad substrate specificity and act on a range of nucleoside di- and triphosphates. In general, NUDIX hydrolases are considered to be “housekeeping” enzymes that maintain a balance between various nucleotide pools.…”