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Panoramic view of a superfamily of phosphatases through substrate profiling

Huang H, Pandya C, Liu C, Al-Obaidi NF, Wang M, Zheng L, Toews-Keating S, Aono M, Love JD, Evan B, Seidel RD, Hillerich BS, Garforth SJ, Almo SC, Mariano PS, Dunaway-Mariano D, Allen KN, Farelli JD (2015) Proc Natl Acad Sci USA, 112, E1974-1983. PMCID: PM

A major high throughput screening endeavor from the HAD Bridging Project and Protein Core has allowed annotations for ~35% of the massive Haloalkanoate Dehalogenase Superfamily. Substrate specificities determined for >200 enzymes paint a detailed picture of the structure-function relationship contributed by a variable cap domain.


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 iso-functional 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.

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Figure Figure 1. Pie chart showing the chemical composition of the substrate library, separated by designated chemical class of the leaving group. Sections are color-coded with corresponding representative structures and percent occurrence is labeled in a like color.

Figure Figure 2. Representative SSN of the HADSF; 79,778 HADSF sequences from the SFLD organized into representative SSNs.

Figure 3. Data from screening of the HADSF.

Figure Figure 4. Heat maps showing activity profiles for screened enzymes.

Figure Figure 5. Functional Annotation of the HADSF by SSN expansion. Individual SFLD subgroups were expanded to annotate function based on either high-throughput screening or information from previously characterized enzymes.

Reprinted with permission from Huang et al. PNAS Copyright © 2015 National Academy of Sciences