Limitations of Current Machine-Learning Models in Predicting Enzymatic Functions for Uncharacterized Proteins.

Unlabelled: Thirty to seventy percent of proteins in any given genome have no assigned function and have been labeled as the protein "unknome". This large knowledge gap prevents the biological community from fully leveraging the plethora of genomic data that is now available. Machine-learning approaches are showing some promise in propagating functional knowledge from experimentally characterized proteins to the correct set of isofunctional orthologs.

On the necessity to include multiple types of evidence when predicting molecular function of proteins.

Machine learning-based platforms are currently revolutionizing many fields of molecular biology including structure prediction for monomers or complexes, predicting the consequences of mutations, or predicting the functions of proteins. However, these platforms use training sets based on currently available knowledge and, in essence, are not built to discover novelty. Hence, claims of discovering novel functions for protein families using artificial intelligence should be carefully dissected, as the dangers of overpredictions are real as we show in a detailed analysis of the prediction made by Kim et al on the function of the YciO protein in the model organism .

7-Deazaguanines in DNA: functional and structural elucidation of a DNA modification system.

The modified nucleosides 2'-deoxy-7-cyano- and 2'-deoxy-7-amido-7-deazaguanosine (dPreQ0 and dADG, respectively) recently discovered in DNA are the products of the bacterial queuosine tRNA modification pathway and the dpd gene cluster, the latter of which encodes proteins that comprise the elaborate Dpd restriction-modification system present in diverse bacteria. Recent genetic studies implicated the dpdA, dpdB and dpdC genes as encoding proteins necessary for DNA modification, with dpdD-dpdK contributing to the restriction phenotype. Here we report the in vitro reconstitution of the Dpd modification machinery from Salmonella enterica serovar Montevideo, the elucidation of the roles of each protein and the X-ray crystal structure of DpdA supported by small-angle X-ray scattering analysis of DpdA and DpdB, the former bound to DNA.

Structural basis of Qng1-mediated salvage of the micronutrient queuine from queuosine-5'-monophosphate as the biological substrate.

Eukaryotic life benefits from-and ofttimes critically relies upon-the de novo biosynthesis and supply of vitamins and micronutrients from bacteria. The micronutrient queuosine (Q), derived from diet and/or the gut microbiome, is used as a source of the nucleobase queuine, which once incorporated into the anticodon of tRNA contributes to translational efficiency and accuracy. Here, we report high-resolution, substrate-bound crystal structures of the Sphaerobacter thermophilus queuine salvage protein Qng1 (formerly DUF2419) and of its human ortholog QNG1 (C9orf64), which together with biochemical and genetic evidence demonstrate its function as the hydrolase releasing queuine from queuosine-5'-monophosphate as the biological substrate.

Predicting the capsid architecture of phages from metagenomic data.

Tailed phages are viruses that infect bacteria and are the most abundant biological entities on Earth. Their ecological, evolutionary, and biogeochemical roles in the planet stem from their genomic diversity. Known tailed phage genomes range from 10 to 735 kilobase pairs thanks to the size variability of the protective protein capsids that store them.

Detection of preQ0 deazaguanine modifications in bacteriophage CAjan DNA using Nanopore sequencing reveals same hypermodification at two distinct DNA motifs.

In the constant evolutionary battle against mobile genetic elements (MGEs), bacteria have developed several defense mechanisms, some of which target the incoming, foreign nucleic acids e.g. restriction-modification (R-M) or CRISPR-Cas systems.

Specificity in the biosynthesis of the universal tRNA nucleoside -threonylcarbamoyl adenosine (tA)-TsaD is the gatekeeper.

-threonylcarbamoyl adenosine (tA) is a nucleoside modification found in all kingdoms of life at position 37 of tRNAs decoding ANN codons, which functions in part to restrict translation initiation to AUG and suppress frameshifting at tandem ANN codons. In Bacteria the proteins TsaB, TsaC (or C2), TsaD, and TsaE, comprise the biosynthetic apparatus responsible for tA formation. TsaC(C2) and TsaD harbor the relevant active sites, with TsaC(C2) catalyzing the formation of the intermediate threonylcarbamoyladenosine monophosphate (TC-AMP) from ATP, threonine, and CO, and TsaD catalyzing the transfer of the threonylcarbamoyl moiety from TC-AMP to A of substrate tRNAs.

Structure-based design of guanosine analogue inhibitors targeting GTP cyclohydrolase IB towards a new class of antibiotics.

GTP cyclohydrolase (GCYH-I) is an enzyme in the folate biosynthesis pathway that has not been previously exploited as an antibiotic target, although several pathogens including N. gonorrhoeae use a form of the enzyme GCYH-IB that is structurally distinct from the human homologue GCYH-IA. A comparison of the crystal structures of GCYH-IA and -IB with the nM inhibitor 8-oxo-GTP bound shows that the active site of GCYH-IB is larger and differently shaped.

Discovery of novel bacterial queuine salvage enzymes and pathways in human pathogens.

Queuosine (Q) is a complex tRNA modification widespread in eukaryotes and bacteria that contributes to the efficiency and accuracy of protein synthesis. Eukaryotes are not capable of Q synthesis and rely on salvage of the queuine base (q) as a Q precursor. While many bacteria are capable of Q de novo synthesis, salvage of the prokaryotic Q precursors preQ and preQ also occurs.

Conformational communication mediates the reset step in t6A biosynthesis.

The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC-AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.

Inhibitor potency varies widely among tumor-relevant human isocitrate dehydrogenase 1 mutants.

Mutations in isocitrate dehydrogenase 1 (IDH1) drive most low-grade gliomas and secondary glioblastomas and many chondrosarcomas and acute myeloid leukemia cases. Most tumor-relevant IDH1 mutations are deficient in the normal oxidization of isocitrate to α-ketoglutarate (αKG), but gain the neomorphic activity of reducing αKG to D-2-hydroxyglutarate (D2HG), which drives tumorigenesis. We found previously that IDH1 mutants exhibit one of two reactivities: deficient αKG and moderate D2HG production (including commonly observed R132H and R132C) or moderate αKG and high D2HG production (R132Q).

Structure and mechanism of a bacterial t6A biosynthesis system.

The universal N(6)-threonylcarbamoyladenosine (t6A) modification at position 37 of ANN-decoding tRNAs is central to translational fidelity. In bacteria, t6A biosynthesis is catalyzed by the proteins TsaB, TsaC/TsaC2, TsaD and TsaE. Despite intense research, the molecular mechanisms underlying t6A biosynthesis are poorly understood.

Protection of the Queuosine Biosynthesis Enzyme QueF from Irreversible Oxidation by a Conserved Intramolecular Disulfide.

QueF enzymes catalyze the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of the nitrile group of 7-cyano-7-deazaguanine (preQ₀) to 7-aminomethyl-7-deazaguanine (preQ₁) in the biosynthetic pathway to the tRNA modified nucleoside queuosine. The QueF-catalyzed reaction includes formation of a covalent thioimide intermediate with a conserved active site cysteine that is prone to oxidation in vivo. Here, we report the crystal structure of a mutant of QueF, which reveals an unanticipated intramolecular disulfide formed between the catalytic Cys55 and a conserved Cys99 located near the active site.

Mechanism and catalytic strategy of the prokaryotic-specific GTP cyclohydrolase-IB.

Guanosine 5'-triphosphate (GTP) cyclohydrolase-I (GCYH-I) catalyzes the first step in folic acid biosynthesis in bacteria and plants, biopterin biosynthesis in mammals, and the biosynthesis of 7-deazaguanosine-modified tRNA nucleosides in bacteria and archaea. The type IB GCYH (GCYH-IB) is a prokaryotic-specific enzyme found in many pathogens. GCYH-IB is structurally distinct from the canonical type IA GCYH involved in biopterin biosynthesis in humans and animals, and thus is of interest as a potential antibacterial drug target.

Crystal structure of the archaeosine synthase QueF-like-Insights into amidino transfer and tRNA recognition by the tunnel fold.

The tunneling-fold (T-fold) structural superfamily has emerged as a versatile protein scaffold of diverse catalytic activities. This is especially evident in the pathways to the 7-deazaguanosine modified nucleosides of tRNA queuosine and archaeosine. Four members of the T-fold superfamily have been confirmed in these pathways and here we report the crystal structure of a fifth enzyme; the recently discovered amidinotransferase QueF-Like (QueF-L), responsible for the final step in the biosynthesis of archaeosine in the D-loop of tRNA in a subset of Crenarchaeota.

Fluorescent fatty acid transfer from bovine serum albumin to phospholipid vesicles: collision or diffusion mediated uptake.

Purpose: The extent of palmitate uptake by hepatocytes is dependent upon the surface charge of the extracellular binding protein. Specifically, hepatocyte uptake is greater when palmitate is bound to cationic binding proteins than when it is bound to anionic proteins. To further understand the role of protein surface charge on the uptake process of protein-bound ligands, we examined the rate of transfer of fluorescent anthroyloxy palmitic acid (AOPA) in the presence of anionic and cationic extracellular proteins to model membranes containing different surface charged groups.

Structural basis of biological nitrile reduction.

The enzyme QueF catalyzes the reduction of the nitrile group of 7-cyano-7-deazaguanine (preQ(0)) to 7-aminomethyl-7-deazaguanine (preQ(1)), the only nitrile reduction reaction known in biology. We describe here two crystal structures of Bacillus subtilis QueF, one of the wild-type enzyme in complex with the substrate preQ(0), trapped as a covalent thioimide, a putative intermediate in the reaction, and the second of the C55A mutant in complex with the substrate preQ(0) bound noncovalently. The QueF enzyme forms an asymmetric tunnel-fold homodecamer of two head-to-head facing pentameric subunits, harboring 10 active sites at the intersubunit interfaces.

Diversity of archaeosine synthesis in crenarchaeota.

Archaeosine (G(+)) is found at position 15 of many archaeal tRNAs. In Euryarchaeota, the G(+) precursor, 7-cyano-7-deazaguanine (preQ(0)), is inserted into tRNA by tRNA-guanine transglycosylase (arcTGT) before conversion into G(+) by ARChaeosine Synthase (ArcS). However, many Crenarchaeota known to harbor G(+) lack ArcS homologues.

YrdC exhibits properties expected of a subunit for a tRNA threonylcarbamoyl transferase.

The post-transcriptional nucleoside modifications of tRNA's anticodon domain form the loop structure and dynamics required for effective and accurate recognition of synonymous codons. The N(6)-threonylcarbamoyladenosine modification at position 37 (t(6)A(37)), 3'-adjacent to the anticodon, of many tRNA species in all organisms ensures the accurate recognition of ANN codons by increasing codon affinity, enhancing ribosome binding, and maintaining the reading frame. However, biosynthesis of this complex modification is only partially understood.

Discovery and characterization of an amidinotransferase involved in the modification of archaeal tRNA.

The presence of the 7-deazaguanosine derivative archaeosine (G(+)) at position 15 in tRNA is one of the diagnostic molecular characteristics of the Archaea. The biosynthesis of this modified nucleoside is especially complex, involving the initial production of 7-cyano-7-deazaguanine (preQ(0)), an advanced precursor that is produced in a tRNA-independent portion of the biosynthesis, followed by its insertion into the tRNA by the enzyme tRNA-guanine transglycosylase (arcTGT), which replaces the target guanine base yielding preQ(0)-tRNA. The enzymes responsible for the biosynthesis of preQ(0) were recently identified, but the enzyme(s) catalyzing the conversion of preQ(0)-tRNA to G(+)-tRNA have remained elusive.

Zinc-independent folate biosynthesis: genetic, biochemical, and structural investigations reveal new metal dependence for GTP cyclohydrolase IB.

GTP cyclohydrolase I (GCYH-I) is an essential Zn(2+)-dependent enzyme that catalyzes the first step of the de novo folate biosynthetic pathway in bacteria and plants, the 7-deazapurine biosynthetic pathway in Bacteria and Archaea, and the biopterin pathway in mammals. We recently reported the discovery of a new prokaryotic-specific GCYH-I (GCYH-IB) that displays no sequence identity to the canonical enzyme and is present in approximately 25% of bacteria, the majority of which lack the canonical GCYH-I (renamed GCYH-IA). Genomic and genetic analyses indicate that in those organisms possessing both enzymes, e.

The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA.

Threonylcarbamoyladenosine (t(6)A) is a universal modification found at position 37 of ANN decoding tRNAs, which imparts a unique structure to the anticodon loop enhancing its binding to ribosomes in vitro. Using a combination of bioinformatic, genetic, structural and biochemical approaches, the universal protein family YrdC/Sua5 (COG0009) was shown to be involved in the biosynthesis of this hypermodified base. Contradictory reports on the essentiality of both the yrdC wild-type gene of Escherichia coli and the SUA5 wild-type gene of Saccharomyces cerevisiae led us to reconstruct null alleles for both genes and prove that yrdC is essential in E.

Discovery of a new prokaryotic type I GTP cyclohydrolase family.

GTP cyclohydrolase I (GCYH-I) is the first enzyme of the de novo tetrahydrofolate biosynthetic pathway present in bacteria, fungi, and plants, and encoded in Escherichia coli by the folE gene. It is also the first enzyme of the biopterin (BH4) pathway in Homo sapiens, where it is encoded by a homologous folE gene. A homology-based search of GCYH-I orthologs in all sequenced bacteria revealed a group of microbes, including several clinically important pathogens, that encoded all of the enzymes of the tetrahydrofolate biosynthesis pathway but GCYH-I, suggesting that an alternate family was present in these organisms.

Two conformations of a crystalline human tRNA synthetase-tRNA complex: implications for protein synthesis.

Aminoacylation of tRNA is the first step of protein synthesis. Here, we report the co-crystal structure of human tryptophanyl-tRNA synthetase and tRNATrp. This enzyme is reported to interact directly with elongation factor 1alpha, which carries charged tRNA to the ribosome.

Crystallization and preliminary X-ray characterization of the nitrile reductase QueF: a queuosine-biosynthesis enzyme.

QueF (MW = 19.4 kDa) is a recently characterized nitrile oxidoreductase which catalyzes the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of the modified tRNA nucleoside queuosine. Initial crystals of homododecameric Bacillus subtilis QueF diffracted poorly to 8.