Enzyme genomics: Application of general enzymatic screens to discover new enzymes

Abstract

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.

A Conserved Carboxylesterase Is a SUPPRESSOR OF AVRBST-ELICITED RESISTANCE inArabidopsis

“…Such assays monitor the formation of p-nitrophenol resulting from the hydrolysis of synthetic p-nitrophenyl ester substrates (Kuznetsova et al, 2005). Both His 6 -SOBER1 and His 6 -ScAPT1 recombinant proteins were able to cleave p-nitrophenyl acetate (C 2 acyl chain); however, SOBER1 specific activity was approximately twofold lower (Figure 8).…”

The crystal structure of the outer membrane lipoprotein YbhC from Escherichia coli sheds new light on the phylogeny of carbohydrate esterase family 8

“…On the basis of the sequence similarity reflected in its CE8 grouping, YbhC has previously been annotated as a PME or, alternately, as a palmitoyl-CoA thioesterase, the latter based on results from a high-throughput enzyme activity screen. 5 Interestingly, phylogenetic analysis indicates that E. coli YbhC and homologs from other gram-negative Enterobacteriaceae form a distinct clade in CE8, which is well-separated from those containing demonstrated bacterial PMEs. This observation, coupled with the ambiguous functional assignment of YbhC, motivated the solution of the tertiary structure of YbhC and a re-examination of its enzyme activity in this context.…”

Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae

“…The proteins were affinity-purified to over 95% homogeneity and first analyzed for Mg 2ϩ -dependent phosphatase activity using the generic phosphatase substrate p-nitrophenyl phosphate (pNPP) (21). Except for YKL033W, most yeast HADs showed readily detectable Mg 2ϩ -dependent hydrolysis of pNPP (Table 1), with the highest activity observed for PHO13 (ϳ200 mol min Ϫ1 mg Ϫ1 protein), which has been annotated as a pNPP phosphatase (72).…”

“…To infer gene function, complementary computational and experimental approaches and their combinations have been used, including sequence analysis, comparative genomics, gene expression and disruption, protein interaction, and protein structure, but ultimately all depend on experimental testing (3,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Recently, the Computational Bridge to Experiments (COMBREX) consortium coordinated a community of computational and experimental scientists to generate functional predictions for the most interesting families of unknown proteins and to carry out experimental characterization (22).…”