Enzyme genomics: Application of general enzymatic screens to discover new enzymes.
In all sequenced genomes, a large fraction of predicted genes encodes proteins of unknown biochemical function and up to 15% of the genes with "known" function are mis-annotated. Several global approaches are routinely employed to predict function, including sophisticated sequence analysis, gene expression, protein interaction, and protein structure. In the first coupling of genomics and enzymology, Phizicky and colleagues undertook a screen for specific enzymes using large pools of partially purified proteins and specific enzymatic assays. Here we present an overview of the further developments of this approach, which involve the use of general enzymatic assays to screen individually purified proteins for enzymatic activity. The assays have relaxed substrate specificity and are designed to identify the subclass or sub-subclasses of enzymes (phosphatase, phosphodiesterase/nuclease, protease, esterase, dehydrogenase, and oxidase) to which the unknown protein belongs. Further biochemical characterization of proteins can be facilitated by the application of secondary screens with natural substrates (substrate profiling). We demonstrate here the feasibility and merits of this approach for hydrolases and oxidoreductases, two very broad and important classes of enzymes. Application of general enzymatic screens and substrate profiling can greatly speed up the identification of biochemical function of unknown proteins and the experimental verification of functional predictions produced by other functional genomics approaches.
Kuznetsova
Ekaterina
E
Banting and Best Department of Medical Research, 112 College St., University of Toronto, Toronto, Ont. M5G 1L6, Canada.
Proudfoot
Michael
M
Sanders
Stephen A
SA
Reinking
Jeffrey
J
Savchenko
Alexei
A
Arrowsmith
Cheryl H
CH
Edwards
Aled M
AM
Yakunin
Alexander F
AF
D004798:Enzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D023281:Genomics / Q000379:methods*; D006801:Humans; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D040901:Proteomics; D013379:Substrate Specificity
D004798:Enzymes; D011506:Proteins; D010088:Oxidoreductases; D006867:Hydrolases