Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family.
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 kcat/Kmvalues >10,000 M-1s-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 kcat/Kmvalues exhibited against these canonical substrates are well under 105M-1s-1. By contrast, several Nudix enzymes show much larger kcat/Kmvalues (in the range of 105to >107M-1s-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 Wiley Periodicals, Inc.
Molecular and Cell Biology Department, University of California, Berkeley, California, 94720.
Vi N
VN
Nguyen
Molecular and Cell Biology Department, University of California, Berkeley, California, 94720.
Annsea
A
Park
Graduate Program in Comparative Biochemistry, University of California, Berkeley, California, 94720.
Anting
A
Xu
Molecular and Cell Biology Department, University of California, Berkeley, California, 94720.|Plant and Microbial Biology Department, University of California, Berkeley, California, 94720.
John R
JR
Srouji
Molecular and Cell Biology Department, University of California, Berkeley, California, 94720.|Graduate Program in Comparative Biochemistry, University of California, Berkeley, California, 94720.|Plant and Microbial Biology Department, University of California, Berkeley, California, 94720.
Steven E
SE
Brenner
Molecular and Cell Biology Department, University of California, Berkeley, California, 94720.|Graduate Program in Comparative Biochemistry, University of California, Berkeley, California, 94720.
Jack F
JF
Kirsch
D000246:Adenosine Diphosphate Ribose / Q000737:chemistry / Q000378:metabolism; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000737:chemistry / Q000201:enzymology; D003838:Deoxyadenine Nucleotides / Q000737:chemistry / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000737:chemistry / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008087:Listeria / Q000737:chemistry / Q000201:enzymology; D005810:Multigene Family; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D000096964:Nudix Hydrolases
D000246:Adenosine Diphosphate Ribose; D001426:Bacterial Proteins; D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D015226:Dinucleoside Phosphates; D011755:Pyrophosphatases; D011994:Recombinant Proteins; D000096964:Nudix Hydrolases; C089930:8-oxodeoxyadenosine triphosphate; C029603:deoxyguanosine triphosphate
Nudix; kinetics; physiological substrate; substrate screening