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.

Diglycine Enables Rapid Intrabacterial Hydrolysis for Activating Anbiotics against Gram‐negative Bacteria

“…[7] Despite its potent activity against Gram-negative bacteria, the clinical use of CL is rather limited because of its adverse effects,s uch as bone marrow suppression. [11] Based on the above rationale,weconjugated the simplest dipeptide,d iglycine (GG), with CLsu to create an ew conjugate CLsuGG.B esides exhibiting about 10 times higher inhibitory activity than CLsu against E. coli,CLsuGG shows lower cytotoxicity than CL towards bone marrow stromal cells.M echanistic studies confirm that ap rotoncoupled and bacterial specific oligopeptide transporter, YdgR, [12] is involved in the uptake of CLsuGG.M ore importantly,CLsuGG is rapidly hydrolyzed by intrabacterial esterases (for example,BioH [13] and YjfP [14] )toregenerate CL (Scheme 1). [10] One strategy to address the problem is to enhance the uptake and increase the retention time of CL inside the bacteria by conjugating it with ad ipeptide because Gram-negative bacteria express oligopeptide transporters for the uptake of di-and tripeptides.…”

The Crystallization Expert System Xtaldb, and its Application to the Structure of the 5'- nucleotidase YfbR and Other Proteins

“…A.3 Crystal structure of pNPP-Na pNPP has been used for decades as a marker of phosphatase activity, as described in Chapter 4. pNPP was originally employed clinically in the detection of alkaline phosphatase activity in blood serum (Bessey et al, 1946) and is used today as a general indicator of phosphohydrolase activity. The anion is a component of a set of general enzymatic screens against libraries of proteins of unknown function (Kuznetsova et al, 2005), and was used to originally determine the function of YfbR as a phosphohydrolase (Chapter 4).…”

“…A number of membraneassociated, periplasmic and extracellular bacterial 5'-NTs have been identified and purified, but few have been extensively characterized, such as UshA from E. coli, NucA from Haemophilus influenzae (Zagursky et al, 2000), or HppA from Helicobacter pylori (Reilly & Calcutt, 2004). Recently, using a systematic general enzymatic screen against a large set of purified bacterial proteins (Kuznetsova et al, 2005), three of the first intracellular bacterial 5'-NTs were identified in E. coli: SurE, YjjG, and YfbR. SurE is a 5'(3')-nucleotidase and a member of a conserved domain found in prokaryotes and eukaryotes.…”

“…Each protein was over-expressed, purified and tested for phosphohydrolase activity against a range of phosphatase and phosphodiesterase substrates (Kuznetsova et al, 2005). In these assays, both AF1432 and YedJ exhibited no significant activity, whereas YfdR was found to be another E. coli 5'-nucleotidase with a substrate range broader than that of YfbR (Fig.…”

“…The general screens were performed essentially as previously described (Kuznetsova et al, 2005). Briefly, purified protein was screened for phosphatase activity using the general phosphatase substrate p-nitrophenyl phosphate ( …”