A machine learning-enabled open biodata resource inventory from the scientific literature.

Modern biological research depends on data resources. These resources archive difficult-to-reproduce data and provide added-value aggregation, curation, and analyses. Collectively, they constitute a global infrastructure of biodata resources.

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

The Enzyme Function Initiative, an NIH/NIGMS-supported Large-Scale Collaborative Project (EFI; U54GM093342; http://enzymefunction.org/), is focused on devising and disseminating bioinformatics and computational tools as well as experimental strategies for the prediction and assignment of functions (in vitro activities and in vivo physiological/metabolic roles) to uncharacterized enzymes discovered in genome projects. Protein sequence similarity networks (SSNs) are visually powerful tools for analyzing sequence relationships in protein families (H.

The Pseudomonas aeruginosa antimetabolite L -2-amino-4-methoxy-trans-3-butenoic acid (AMB) is made from glutamate and two alanine residues via a thiotemplate-linked tripeptide precursor.

The Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is a non-proteinogenic amino acid which is toxic for prokaryotes and eukaryotes. Production of AMB requires a five-gene cluster encoding a putative LysE-type transporter (AmbA), two non-ribosomal peptide synthetases (AmbB and AmbE), and two iron(II)/α-ketoglutarate-dependent oxygenases (AmbC and AmbD). Bioinformatics analysis predicts one thiolation (T) domain for AmbB and two T domains (T1 and T2) for AmbE, suggesting that AMB is generated by a processing step from a precursor tripeptide assembled on a thiotemplate.

Mechanistic diversity in the RuBisCO superfamily: RuBisCO from Rhodospirillum rubrum is not promiscuous for reactions catalyzed by RuBisCO-like proteins.

d-Ribulose 1,5-bisphosphate carboxylase/oxygenases (RuBisCOs) are promiscuous, catalyzing not only carboxylation and oxygenation of d-ribulose 1,5-bisphosphate but also other promiscuous, presumably nonphysiological, reactions initiated by abstraction of the 3-proton of d-ribulose 1,5-bisphosphate. Also, RuBisCO has homologues that do not catalyze carboxylation; these are designated RuBisCO-like proteins or RLPs. Members of the two families of RLPs catalyze reactions in the recycling of 5'-methylthioadenosine (MTA) generated by polyamine synthesis: (1) the 2,3-diketo-5-methylthiopentane 1-phosphate (DK-MTP 1-P) "enolase" reaction in the well-known "methionine salvage" pathway in Bacillus sp.

A RubisCO-like protein links SAM metabolism with isoprenoid biosynthesis.

Functional assignment of uncharacterized proteins is a challenge in the era of large-scale genome sequencing. Here, we combine in extracto NMR, proteomics and transcriptomics with a newly developed (knock-out) metabolomics platform to determine a potential physiological role for a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-like protein from Rhodospirillum rubrum. Our studies unraveled an unexpected link in bacterial central carbon metabolism between S-adenosylmethionine-dependent polyamine metabolism and isoprenoid biosynthesis and also provide an alternative approach to assign enzyme function at the organismic level.

1-methylthio-D-xylulose 5-phosphate methylsulfurylase: a novel route to 1-deoxy-D-xylulose 5-phosphate in Rhodospirillum rubrum.

Rhodospirillum rubrum produces 5-methylthioadenosine (MTA) from S-adenosylmethionine in polyamine biosynthesis; however, R. rubrum lacks the classical methionine salvage pathway. Instead, MTA is converted to 5-methylthio-d-ribose 1-phosphate (MTR 1-P) and adenine; MTR 1-P is isomerized to 1-methylthio-d-xylulose 5-phosphate (MTXu 5-P) and reductively dethiomethylated to 1-deoxy-d-xylulose 5-phosphate (DXP), an intermediate in the nonmevalonate isoprenoid pathway [Erb, T.

Homology models guide discovery of diverse enzyme specificities among dipeptide epimerases in the enolase superfamily.

The rapid advance in genome sequencing presents substantial challenges for protein functional assignment, with half or more of new protein sequences inferred from these genomes having uncertain assignments. The assignment of enzyme function in functionally diverse superfamilies represents a particular challenge, which we address through a combination of computational predictions, enzymology, and structural biology. Here we describe the results of a focused investigation of a group of enzymes in the enolase superfamily that are involved in epimerizing dipeptides.

The Enzyme Function Initiative.

The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic, we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities.

N-acylation during glidobactin biosynthesis by the tridomain nonribosomal peptide synthetase module GlbF.

Glidobactins are hybrid NRPS-PKS natural products that function as irreversible proteasome inhibitors. A variety of medium chain 2(E),4(E)-diene fatty acids N-acylate the peptidolactam core and contribute significantly to the potency of proteasome inhibition. We have expressed the initiation NRPS module GlbF (C-A-T) in Escherichia coli and observe soluble active protein only on coexpression with the 8 kDa MbtH-like protein, GlbE.

Activation of the pacidamycin PacL adenylation domain by MbtH-like proteins.

Nonribosomal peptide synthetase (NRPS) assembly lines are major avenues for the biosynthesis of a vast array of peptidyl natural products. Several hundred bacterial NRPS gene clusters contain a small (∼70-residue) protein belonging to the MbtH family for which no function has been defined. Here we show that two strictly conserved Trp residues in MbtH-like proteins contribute to stimulation of amino acid adenylation in some NRPS modules.

SylC catalyzes ureido-bond formation during biosynthesis of the proteasome inhibitor syringolin A.

Syringolins are a class of cyclic tripeptide natural products that are potent and irreversible inhibitors of the eukaryotic proteasome. In addition to being hybrid NRPS/PKS molecules, they also feature an unusual ureido-linkage (red) between two amino acid monomers. Here we report the first in vitro characterization of enzymatic ureido-linkage formation which is catalyzed by an NRPS, SylC.

Mechanism of the orotidine 5'-monophosphate decarboxylase-catalyzed reaction: evidence for substrate destabilization.

The reaction catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC) involves a stabilized anionic intermediate, although the structural basis for the rate acceleration (k(cat)/k(non), 7.1 x 10(16)) and proficiency [(k(cat)/K(M))/k(non), 4.8 x 10(22) M(-1)] is uncertain.

Discovery of a dipeptide epimerase enzymatic function guided by homology modeling and virtual screening.

We have developed a computational approach to aid the assignment of enzymatic function for uncharacterized proteins that uses homology modeling to predict the structure of the binding site and in silico docking to identify potential substrates. We apply this method to proteins in the functionally diverse enolase superfamily that are homologous to the characterized L-Ala-D/L-Glu epimerase from Bacillus subtilis. In particular, a protein from Thermotoga martima was predicted to have different substrate specificity, which suggests that it has a different, but as yet unknown, biological function.

Mechanistic diversity in the RuBisCO superfamily: a novel isomerization reaction catalyzed by the RuBisCO-like protein from Rhodospirillum rubrum.

Some homologues of D-ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) do not catalyze carboxylation and are designated RuBisCO-like proteins (RLPs). The RLP from Rhodospirillum rubrum (gi:83593333) catalyzes a novel isomerization reaction (overall 1,3-proton transfer reaction; likely, two 1,2-proton transfer reactions) that converts 5-methylthio-D-ribulose 1-phosphate to a 3:1 mixture of 1-methylthioxylulose 5-phosphate and 1-methylthioribulose 5-phosphate. Disruption of the gene encoding the RLP abolishes the ability of R.

Prediction and assignment of function for a divergent N-succinyl amino acid racemase.

The protein databases contain many proteins with unknown function. A computational approach for predicting ligand specificity that requires only the sequence of the unknown protein would be valuable for directing experiment-based assignment of function. We focused on a family of unknown proteins in the mechanistically diverse enolase superfamily and used two approaches to assign function: (i) enzymatic assays using libraries of potential substrates, and (ii) in silico docking of the same libraries using a homology model based on the most similar (35% sequence identity) characterized protein.

A disaccharide rearrangement catalyzed by molybdate anion in aqueous solution.

Reaction of a galactosylated 2-C-(hydroxymethyl)-tetrofuranose with paramolybdate ion-exchange resin in aqueous solution at 67 degrees C gave an equililibrium mixture containing the reactant aldofuranose (42%) and a 2-ketopentofuranose galactosylated at O1 (58%). Observation of this stereospecific rearrangement supports prior arguments that substituents at C2 of the branched-chain aldofuranose reactant are located in a sterically accessible pocket of the putative dimolybdate-saccharide reactive complex during epimerization. This rearrangement provides a new and convenient route to 2-ketosugars glycosylated at the exocyclic C1 position.

Mechanistic diversity in the RuBisCO superfamily: the "enolase" in the methionine salvage pathway in Geobacillus kaustophilus.

D-Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme, is the paradigm member of the recently recognized mechanistically diverse RuBisCO superfamily. The RuBisCO reaction is initiated by abstraction of the proton from C3 of the d-ribulose 1,5-bisphosphate substrate by a carbamate oxygen of carboxylated Lys 201 (spinach enzyme). Heterofunctional homologues of RuBisCO found in species of Bacilli catalyze the tautomerization ("enolization") of 2,3-diketo-5-methylthiopentane 1-phosphate (DK-MTP 1-P) in the methionine salvage pathway in which 5-methylthio-d-ribose (MTR) derived from 5'-methylthioadenosine is converted to methionine [Ashida, H.