Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST): A web tool for generating protein sequence similarity networks

Gerlt, J.A.; Bouvier, Jason T.; Davidson, Daniel B.; Imker, H.J.; Sadkhin, Boris; Slater, David R.; Whalen, K.L.

doi:10.1016/j.bbapap.2015.04.015

Cited by 717 publications

(806 citation statements)

References 53 publications

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“…The protein association network analysis was performed on the STRING database (http://string-db.org/) (Szklarczyk et al, 2015). The Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST) (http://enzymefunction.org/) was used to extract a physical clustering network (Gerlt et al, 2015) as follows. The amino acid sequences of proteins of the IPR011078 InterPro family were extracted to generate a sequence similarity network with an original alignment score threshold of 30 and no restrictions for alignment lengths.…”

Section: Methodsmentioning

confidence: 99%

Evidence that COG0325 proteins are involved in PLP homeostasis

et al. 2016

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Pyridoxal 59-phosphate (PLP) is an essential cofactor for nearly 60 Escherichia coli enzymes but is a highly reactive molecule that is toxic in its free form. How PLP levels are regulated and how PLP is delivered to target enzymes are still open questions. The COG0325 protein family belongs to the fold-type III class of PLP enzymes and binds PLP but has no known biochemical activity although it occurs in all kingdoms of life. Various pleiotropic phenotypes of the E. coli COG0325 (yggS) mutant have been reported, some of which were reproduced and extended in this study. Comparative genomic, genetic and metabolic analyses suggest that these phenotypes reflect an imbalance in PLP homeostasis. The E. coli yggS mutant accumulates the PLP precursor pyridoxine 59-phosphate (PNP) and is sensitive to an excess of pyridoxine but not of pyridoxal. The pyridoxine toxicity phenotype is complemented by the expression of eukaryotic yggS orthologs. It is also suppressed by the presence of amino acids, specifically isoleucine, threonine and leucine, suggesting the PLP-dependent enzyme transaminase B (IlvE) is affected. These genetic results lay a foundation for future biochemical studies of the role of COG0325 proteins in PLP homeostasis.

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Section: Methodsmentioning

confidence: 99%

Evidence that COG0325 proteins are involved in PLP homeostasis

et al. 2016

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“…Analysis of Sequence Similarity Network (SSN)-A sequence similarity network (SSN) for HadA was carried out using the option A of Enzyme Function Initiative (EFI)-Enzyme Similarity Tool and sequences from UniProt database that had identity to HadA of 34% or higher (63,64). SSNs of HadA and its homologs were generated using an EFI alignment score of 70 (equivalent to grouping sequences of Ͼ27.74% in the same cluster).…”

Section: Identification Of Products From Single Turnovermentioning

confidence: 99%

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

Pimviriyakul

Thotsaporn

Sucharitakul

et al. 2017

Journal of Biological Chemistry

108

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Edited by F. Peter GuengerichThe accumulation of chlorophenols (CPs) in the environment, due to their wide use as agrochemicals, has become a serious environmental problem. These organic halides can be degraded by aerobic microorganisms, where the initial steps of various biodegradation pathways include an oxidative dechlorinating process in which chloride is replaced by a hydroxyl substituent. Harnessing these dechlorinating processes could provide an opportunity for environmental remediation, but detailed catalytic mechanisms for these enzymes are not yet known. To close this gap, we now report transient kinetics and product analysis of the dechlorinating flavin-dependent monooxygenase, HadA, from the aerobic organism Ralstonia pickettii DTP0602, identifying several mechanistic properties that differ from other enzymes in the same class. We first overexpressed and purified HadA to homogeneity. Analyses of the products from single and multiple turnover reactions demonstrated that HadA prefers 4-CP and 2-CP over CPs with multiple substituents. Stopped-flow and rapid-quench flow experiments of HadA with 4-CP show the involvement of specific intermediates (C4a-hydroperoxy-FAD and C4a-hydroxy-FAD) in the reaction, define rate constants and the order of substrate binding, and demonstrate that the hydroxylation step occurs prior to chloride elimination. The data also identify the non-productive and productive paths of the HadA reactions and demonstrate that product formation is the rate-limiting step. This is the first elucidation of the kinetic mechanism of a two-component flavin-dependent monooxygenase that can catalyze oxidative dechlorination of various CPs, and as such it will serve as the basis for future investigation of enzyme variants that will be useful for applications in detoxifying chemicals hazardous to human health.

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“…Taxonomic annotations for the sequences were obtained from the NCBI Taxonomy database and sequences duplicated at the species level were deleted. Clustering on sequence similarity networks (Atkinson et al, 2009) generated using the Enzyme Function-Initiative Enzyme Similarity Tool (Gerlt et al, 2015) were used to identify homologs of characterized proteins from nonspecific hits. In this analysis, nodes represent individual proteins and edges represent the all-versus-all BLAST E-values (Altschul et al, 1990) between them.…”

Section: Gene Sequence Retrievalmentioning

confidence: 99%

The methanogenic redox cofactor F420 is widely synthesized by aerobic soil bacteria

et al. 2016

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F 420 is a low-potential redox cofactor that mediates the transformations of a wide range of complex organic compounds. Considered one of the rarest cofactors in biology, F 420 is best known for its role in methanogenesis and has only been chemically identified in two phyla to date, the Euryarchaeota and Actinobacteria. In this work, we show that this cofactor is more widely distributed than previously reported. We detected the genes encoding all five known F 420 biosynthesis enzymes (cofC, cofD, cofE, cofG and cofH) in at least 653 bacterial and 173 archaeal species, including members of the dominant soil phyla Proteobacteria, Chloroflexi and Firmicutes. Metagenome datamining validated that these genes were disproportionately abundant in aerated soils compared with other ecosystems. We confirmed through high-performance liquid chromatography analysis that aerobically grown stationary-phase cultures of three bacterial species, Paracoccus denitrificans, Oligotropha carboxidovorans and Thermomicrobium roseum, synthesized F 420 , with oligoglutamate sidechains of different lengths. To understand the evolution of F 420 biosynthesis, we also analyzed the distribution, phylogeny and genetic organization of the cof genes. Our data suggest that although the F o precursor to F 420 originated in methanogens, F 420 itself was first synthesized in an ancestral actinobacterium. F 420 biosynthesis genes were then disseminated horizontally to archaea and other bacteria. Together, our findings suggest that the cofactor is more significant in aerobic bacterial metabolism and soil ecosystem composition than previously thought. The cofactor may confer several competitive advantages for aerobic soil bacteria by mediating their central metabolic processes and broadening the range of organic compounds they can synthesize, detoxify and mineralize.

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Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST): A web tool for generating protein sequence similarity networks

Cited by 717 publications

References 53 publications

Evidence that COG0325 proteins are involved in PLP homeostasis

Evidence that COG0325 proteins are involved in PLP homeostasis

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

The methanogenic redox cofactor F420 is widely synthesized by aerobic soil bacteria

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