Panoramic view of a superfamily of phosphatases through substrate profiling

Abstract

Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of substrate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified isofunctional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process.evolution | specificity | phosphatase | substrate screen | promiscuity S ince the first genomes were sequenced, there has been an exponential increase in the number of protein sequences deposited into databases worldwide. At the time of this writing the UniProtKB/TrEMBL database contains over 32 million protein sequences. Although this increase in sequence data has dramatically enhanced our understanding of the genomic organization of organisms, as the number of protein sequences grows, the proportion of firm functional assignments diminishes. Traditionally, methods of functional annotation involve comparing sequence identity between experimentally characterized proteins and newly sequenced ones, typically via BLAST (1). In cases where significant sequence similarity cannot be ascertained, proteins are annotated as "hypothetical" or "putative." Moreover, the decrease in sequence identity leads to an increased uncertainty in functional assignment, especially as the phylogenetic distance between organisms grows, limiting iso-functional ortholog discovery.As the number of newly sequenced genomes grows larger, more protein sequences are likely to be misannotated, oftentimes resulting in the propagation of incorrect functional annotation across newly identified sequences. To tackle the problem of unannotated or misannotated proteins, newer methods for computational assignment have been created with varying degrees of success (2). Although these methods outperform historical methods, continued improvement is necessary to ensure accurate annotation of function (2). A greater swath of functional space can be covered by screening substrates in a high-throughput manner on multiple enzymes from a family (3, 4). Family-wide substrate profiling offers a data-rich resource. The use of sparse screening of sequence space and a diversified library permits the determination of substrate specificity profiles to provide a familywide view of the range of substrates...

Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis

“…This suggests that ysaA may be the B. subitilis equivalent of serB . Accordingly, we examined the phosphohydrolase activity of YsaA in vitro , screening YsaA against a library of 167 phosphorylated substrates with a stop-point colorimetric assay (Huang et al, 2015). YsaA showed phosphatase activity against phosphoserine, phosphothreonine, phosphoethanolamine and histidinol phosphate.…”

“…When the cultures reached OD 600 of 0.4, expression of YsaA was induced by addition of IPTG to a final concentration of 1 mM and growth continued at 30 °C with aeration for 2 hr. Purification of YsaA was carried out as described previously (Huang et al, 2015). Cells were suspended in lysis buffer [20 mM HEPES (pH 7.5), 500 mM NaCl, 20 mM imidazole, and 10% (vol/vol) glycerol] and lysed by sonication.…”

Biology, Mechanism, and Structure of Enzymes in the α- d -Phosphohexomutase Superfamily

“…It should also help correct the widespread functional misannotation of these enzymes in databases. This ambitious goal is now feasible using a combination of approaches such as high-throughput substrate profiling and bioinformatic analyses, as has been done with other enzyme families (Burroughs et al, 2006; Huang et al, 2015; Pandya et al, 2013). …”

Catalytic scaffolds for phosphoryl group transfer

“…It has been pointed out that the large volume, polar solvent accessible surface area and varied electrostatic surface of the active site of the AP superfamily enables binding to diverse substrates [48]. In contrast, in the HAD superfamily, the insertion of active site loops/domains increases the number of possible interactions with different substrates [49,50]. In both cases, the unifying theme is the availability of multiple or physicochemically flexible interaction sites.…”