Prediction and characterization of enzymatic activities guided by sequence similarity and genome neighborhood networks

Zhao, Suwen; Sakai, A.; Zhang, Xinshuai; Vetting, M.W.; Kumar, Ritesh; Hillerich, B.; Francisco, Brian San; Solbiati, José O.; Steves, Adam; Brown, Shoshana D.; Akiva, Eyal; Barber, Alan E.; Seidel, R.D.; Babbitt, Patricia C.; Almo, Steven C.; Gerlt, J.A.; Jacobson, Matthew P.

doi:10.7554/elife.03275

Cited by 86 publications

(102 citation statements)

References 51 publications

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“…expected to be isofunctional and annotations are made by propagation of the assignments from screened enzymes to the subcluster in which they reside, as has been described for the proline racemase superfamily (37). For example, the C1.5.5 subfamily consisting of 1,994 members was previously broadly annotated as a subfamily of heptose bisphosphate phosphate-like phosphatases.…”

Section: Resultsmentioning

confidence: 99%

Panoramic view of a superfamily of phosphatases through substrate profiling

Huang

Pandya

Liu

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

124

139

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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...

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Section: Resultsmentioning

confidence: 99%

Panoramic view of a superfamily of phosphatases through substrate profiling

Huang

Pandya

Liu

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

124

139

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“…1B. Together with other reports (12,25,59,(62)(63)(64), this report defines the genes that encode specific enzyme activities required for trans-4-hydroxy-L-proline catabolism. These studies should allow the identification of hydroxyproline catabolic genes in other organisms where many of these genes are annotated as genes of unknown function.…”

Section: Discussionmentioning

confidence: 57%

l -Hydroxyproline and d -Proline Catabolism in Sinorhizobium meliloti

et al. 2016

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Sinorhizobium meliloti IMPORTANCEHydroxyproline is abundant in proteins in animal and plant tissues and serves as a carbon and a nitrogen source for bacteria in diverse environments, including the rhizosphere, compost, and the mammalian gut. While the main biochemical features of bacterial hydroxyproline catabolism were elucidated in the 1960s, the genetic and molecular details have only recently been determined. Elucidating the genetics of hydroxyproline catabolism will aid in the annotation of these genes in other genomes and metagenomic libraries. This will facilitate an improved understanding of the importance of this pathway and may assist in determining the prevalence of hydroxyproline in a particular environment.4-Hydroxyproline and 3-hydroxyproline in animal and plant proteins are formed through the posttranslational modification of proline residues by the enzyme proline hydroxylase. In some bacteria, hydroxyproline is synthesized from free L-proline, where it is employed in the synthesis of secondary metabolites (1, 2). 4-Hydroxyproline has two chiral carbon atoms, and of its four isomeric forms, trans-4-hydroxy-L-proline (trans-4-L-Hyp) and cis-4-hydroxy-D-proline (cis-4-D-Hyp) appear to be the two most common isomers. trans-4-

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“…We focus on the SBPs of bacterial TRAP (tripartite ATP-independent permease) and ATP-binding cassette transport systems; both have an extracellular SBP that binds and delivers its ligand to the integral membrane permease components for transport into the cell (19,20). We then synergistically use protein family sequence similarity networks (SSNs) and genome neighborhood networks (GNNs) to discover the enzyme components of the pathway and infer their functions (21). A SSN is a readily accessible method (constructed with the Enzyme Function Initiative-Enzyme Similarity Tool web tool) for segregating protein families, including those of SBPs as well as enzymes, into isofunctional groups (22).…”

mentioning

confidence: 99%

Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars

Zhang

Carter

Vetting

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Using a large-scale "genomic enzymology" approach, we (i) assigned novel ATP-dependent four-carbon acid sugar kinase functions to members of the DUF1537 protein family (domain of unknown function; Pfam families PF07005 and PF17042) and (ii) discovered novel catabolic pathways for D-threonate, L-threonate, and D-erythronate. The experimentally determined ligand specificities of several solute binding proteins (SBPs) for TRAP (tripartite ATP-independent permease) transporters for four-carbon acids, including D-erythronate and L-erythronate, were used to constrain the substrates for the catabolic pathways that degrade the SBP ligands to intermediates in central carbon metabolism. Sequence similarity networks and genome neighborhood networks were used to identify the enzyme components of the pathways. Conserved genome neighborhoods encoded SBPs as well as permease components of the TRAP transporters, members of the DUF1537 family, and a member of the 4-hydroxy-L-threonine 4-phosphate dehydrogenase (PdxA) oxidative decarboxylase, class II aldolase, or ribulose 1,5-bisphosphate carboxylase/oxygenase, large subunit (RuBisCO) superfamily. Because the characterized substrates of members of the PdxA, class II aldolase, and RuBisCO superfamilies are phosphorylated, we postulated that the members of the DUF1537 family are novel ATP-dependent kinases that participate in catabolic pathways for four-carbon acid sugars. We determined that (i) the DUF1537/PdxA pair participates in a pathway for the conversion of D-threonate to dihydroxyacetone phosphate and CO 2 and (ii) the DUF1537/class II aldolase pair participates in pathways for the conversion of D-erythronate and L-threonate (epimers at carbon-3) to dihydroxyacetone phosphate and CO 2 . The physiological importance of these pathways was demonstrated in vivo by phenotypic and genetic analyses.DUF1537 | kinase | four-carbon acid sugars | conserved genome neighborhoods | genomic enzymology

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Prediction and characterization of enzymatic activities guided by sequence similarity and genome neighborhood networks

Cited by 86 publications

References 51 publications

Panoramic view of a superfamily of phosphatases through substrate profiling

Panoramic view of a superfamily of phosphatases through substrate profiling

l -Hydroxyproline and d -Proline Catabolism in Sinorhizobium meliloti

Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars

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