NRPS substrate promiscuity diversifies the xenematides.
Ontology highlight
ABSTRACT:
SUBMITTER: Crawford JM
PROVIDER: S-EPMC3184645 | biostudies-literature | 2011 Oct
REPOSITORIES: biostudies-literature
Publications
NRPS substrate promiscuity diversifies the xenematides.
Crawford Jason M JM Portmann Cyril C Kontnik Renee R Walsh Christopher T CT Clardy Jon J
Organic letters 20110902 19
Xenematide, a cyclic depsipeptide antibiotic produced by Xenorhabdus nematophila, had a candidate nonribosomal peptide synthetase (NRPS) with atypical features. Differential metabolite analysis between a mutant and wildtype validated that this stand-alone NRPS was required for xenematide production, and further analysis led to a series of new xenematide derivatives encoded by the same NRPS. Our results indicate that adenylation domain promiscuity and relaxed downstream processing in the X. nemat ...[more]
Similar Datasets
Project description:N-Acetylhexosamine 1-kinase (NahK) catalyzes the direct addition of a phosphate from adenosine 5'-triphosphate (ATP) to the anomeric position of N-acetylhexosamine and shows similar activity towards N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). Herein we report the cloning, characterization, and substrate specificity studies of two NahKs from Bifidobacterium infantis ATCC15697 and Bifidobacterium longum ATCC55813, respectively. A new capillary electrophoresis assay method has been developed for enzyme activity assays. Both enzymes have a good expression level in E. coli (180-185 mg/L culture) and can tolerate diverse modifications at C2 of GlcNAc and GalNAc. Various GlcNAc derivatives with C6, both C2 and C6, as well as both C2 and C3 modifications are tolerable substrates for the newly cloned NahKs. Quite interestingly, despite of their low activities toward glucose and galactose, the activities of both NahKs are much higher for mannose and some of its C2, C4, and C6 derivatives. These NahKs are excellent catalysts for enzymatic and chemoenzymatic synthesis of carbohydrates.
| S-EPMC6264712 | biostudies-literature
Project description:Deoxycytidine kinase (dCK) is an essential nucleoside kinase critical for the production of nucleotide precursors for DNA synthesis. This enzyme catalyzes the initial conversion of the nucleosides deoxyadenosine (dA), deoxyguanosine (dG), and deoxycytidine (dC) into their monophosphate forms, with subsequent phosphorylation to the triphosphate forms performed by additional enzymes. Several nucleoside analog prodrugs are dependent on dCK for their pharmacological activation, and even nucleosides of the non-physiological L-chirality are phosphorylated by dCK. In addition to accepting dC and purine nucleosides (and their analogs) as phosphoryl acceptors, dCK can utilize either ATP or UTP as phosphoryl donors. To unravel the structural basis for substrate promiscuity of dCK at both the nucleoside acceptor and nucleotide donor sites, we solved the crystal structures of the enzyme as ternary complexes with the two enantiomeric forms of dA (D-dA, or L-dA), with either UDP or ADP bound to the donor site. The complexes with UDP revealed an open state of dCK in which the nucleoside, either D-dA or L-dA, is surprisingly bound in a manner not consistent with catalysis. In contrast, the complexes with ADP, with either D-dA or L-dA, adopted a closed and catalytically competent conformation. The differential states adopted by dCK in response to the nature of the nucleotide were also detected by tryptophan fluorescence experiments. Thus, we are in the unique position to observe differential effects at the acceptor site due to the nature of the nucleotide at the donor site, allowing us to rationalize the different kinetic properties observed with UTP to those with ATP.
| S-EPMC2426910 | biostudies-literature
Project description:Secondary metabolites are assembled by enzymes that often perform reactions with high selectivity and specificity. Many of these enzymes also tolerate variations in substrate structure, exhibiting promiscuity that enables various applications of a given biocatalyst. However, initial enzyme characterization studies frequently do not explore beyond the native substrates. This limited assessment of substrate scope contributes to the difficulty of identifying appropriate enzymes for specific synthetic applications. Here, we report the natural function of cyanobacterial SxtG, an amidinotransferase involved in the biosynthesis of paralytic shellfish toxins, and demonstrate its ability to modify a breadth of non-native substrates. In addition, we report the first X-ray crystal structure of SxtG, which provides rationale for this enzyme's substrate scope. Taken together, these data confirm the function of SxtG and exemplify its potential utility in biocatalytic synthesis.
| S-EPMC7362304 | biostudies-literature
Project description:The enzymatic transfer of the sugar mannose from activated sugar donors is central to the synthesis of a wide range of biologically significant polysaccharides and glycoconjugates. In addition to their importance in cellular biology, mannosyltransferases also provide model systems with which to study catalytic mechanisms of glycosyl transfer. Mannosylglycerate synthase (MGS) catalyzes the synthesis of ?-mannosyl-D-glycerate using GDP-mannose as the preferred donor species, a reaction that occurs with a net retention of anomeric configuration. Past work has shown that the Rhodothermus marinus MGS, classified as a GT78 glycosyltransferase, displays a GT-A fold and performs catalysis in a metal ion-dependent manner. MGS shows very unusual metal ion dependences with Mg(2+) and Ca(2+) and, to a lesser extent, Mn(2+), Ni(2+), and Co(2+), thus facilitating catalysis. Here, we probe these dependences through kinetic and calorimetric analyses of wild-type and site-directed variants of the enzyme. Mutation of residues that interact with the guanine base of GDP are correlated with a higher k(cat) value, whereas substitution of His-217, a key component of the metal coordination site, results in a change in metal specificity to Mn(2+). Structural analyses of MGS complexes not only provide insight into metal coordination but also how lactate can function as an alternative acceptor to glycerate. These studies highlight the role of flexible loops in the active center and the subsequent coordination of the divalent metal ion as key factors in MGS catalysis and metal ion dependence. Furthermore, Tyr-220, located on a flexible loop whose conformation is likely influenced by metal binding, also plays a critical role in substrate binding.
| S-EPMC3083222 | biostudies-literature
Project description:MTH1 (NUDT1) is an oncologic target involved in the prevention of DNA damage. We investigate the way MTH1 recognises its substrates and present substrate-bound structures of MTH1 for 8-oxo-dGTP and 8-oxo-rATP as examples of novel strong and weak binding substrate motifs. Investigation of a small set of purine-like fragments using 2D NMR resulted in identification of a fragment with weak potency. The protein-ligand X-Ray structure of this fragment provides insight into the role of water molecules in substrate selectivity. Wider fragment screening by NMR resulted in three new protein structures exhibiting alternative binding configurations to the key Asp-Asp recognition element of the protein. These inhibitor binding modes demonstrate that MTH1 employs an intricate yet promiscuous mechanism of substrate anchoring through its Asp-Asp pharmacophore. The structures suggest that water-mediated interactions convey selectivity towards oxidized substrates over their non-oxidised counterparts, in particular by stabilization of a water molecule in a hydrophobic environment through hydrogen bonding. These findings may be useful in the design of inhibitors of MTH1.
| S-EPMC4801406 | biostudies-literature
Project description:Seryl-tRNA synthetase (SerRS) attaches L-serine to the cognate serine tRNA (tRNASer) and the noncognate selenocysteine tRNA (tRNASec). The latter activity initiates the anabolic cycle of selenocysteine (Sec), proper decoding of an in-frame Sec UGA codon, and synthesis of selenoproteins across all domains of life. While the accuracy of SerRS is important for overall proteome integrity, it is its substrate promiscuity that is vital for the integrity of the selenoproteome. This raises a question as to what elements in the two tRNA species, harboring different anticodon sequences and adopting distinct folds, facilitate aminoacylation by a common aminoacyl-tRNA synthetase. We sought to answer this question by analyzing the ability of human cytosolic SerRS to bind and act on tRNASer, tRNASec, and 10 mutant and chimeric constructs in which elements of tRNASer were transposed onto tRNASec We show that human SerRS only subtly prefers tRNASer to tRNASec, and that discrimination occurs at the level of the serylation reaction. Surprisingly, the tRNA mutants predicted to adopt either the 7/5 or 8/5 fold are poor SerRS substrates. In contrast, shortening of the acceptor arm of tRNASec by a single base pair yields an improved SerRS substrate that adopts an 8/4 fold. We suggest that an optimal tertiary arrangement of structural elements within tRNASec and tRNASer dictate their utility for serylation. We also speculate that the extended acceptor-TΨC arm of tRNASec evolved as a compromise for productive binding to SerRS while remaining the major recognition element for other enzymes involved in Sec and selenoprotein synthesis.
| S-EPMC5648036 | biostudies-literature
Project description:Minimalist enzymes designed to catalyze model reactions provide useful starting points for creating catalysts for practically important chemical transformations. We have shown that Kemp eliminases of the AlleyCat family facilitate conversion of leflunomide (an immunosupressor pro-drug) to its active form teriflunomide with outstanding rate enhancement (nearly four orders of magnitude) and catalytic proficiency (more than seven orders of magnitude) without any additional optimization. This remarkable activity is achieved by properly positioning the substrate in close proximity to the catalytic glutamate with very high pKa.
| S-EPMC6884320 | biostudies-literature
Project description:The ASCE (additional strand, conserved E) superfamily of proteins consists of structurally similar ATPases associated with diverse cellular activities involving metabolism and transport of proteins and nucleic acids in all forms of life. A subset of these enzymes consists of multimeric ringed pumps responsible for DNA transport in processes including genome packaging in adenoviruses, herpesviruses, poxviruses and tailed bacteriophages. Although their mechanism of mechanochemical conversion is beginning to be understood, little is known about how these motors engage their nucleic acid substrates. Questions remain as to whether the motors contact a single DNA element, such as a phosphate or a base, or whether contacts are distributed over several parts of the DNA. Furthermore, the role of these contacts in the mechanochemical cycle is unknown. Here we use the genome packaging motor of the Bacillus subtilis bacteriophage varphi29 (ref. 4) to address these questions. The full mechanochemical cycle of the motor, in which the ATPase is a pentameric-ring of gene product 16 (gp16), involves two phases-an ATP-loading dwell followed by a translocation burst of four 2.5-base-pair (bp) steps triggered by hydrolysis product release. By challenging the motor with a variety of modified DNA substrates, we show that during the dwell phase important contacts are made with adjacent phosphates every 10-bp on the 5'-3' strand in the direction of packaging. As well as providing stable, long-lived contacts, these phosphate interactions also regulate the chemical cycle. In contrast, during the burst phase, we find that DNA translocation is driven against large forces by extensive contacts, some of which are not specific to the chemical moieties of DNA. Such promiscuous, nonspecific contacts may reflect common translocase-substrate interactions for both the nucleic acid and protein translocases of the ASCE superfamily.
| S-EPMC2769991 | biostudies-literature
Project description:Mass spectrometry coupled with chromatography separation techniques provides a powerful platform for untargeted metabolomics. Determining the chemical identities of detected compounds however remains a major challenge. Here, we present a novel computational workflow, termed extended metabolic model filtering (EMMF), that aims to engineer a candidate set, a listing of putative chemical identities to be used during annotation, through an extended metabolic model (EMM). An EMM includes not only canonical substrates and products of enzymes already cataloged in a database through a reference metabolic model, but also metabolites that can form due to substrate promiscuity. EMMF aims to strike a balance between discovering previously uncharacterized metabolites and the computational burden of annotation. EMMF was applied to untargeted LC-MS data collected from cultures of Chinese hamster ovary (CHO) cells and murine cecal microbiota. EMM metabolites matched, on average, to 23.92% of measured masses, providing a > 7-fold increase in the candidate set size when compared to a reference metabolic model. Many metabolites suggested by EMMF are not catalogued in PubChem. For the CHO cell, we experimentally confirmed the presence of 4-hydroxyphenyllactate, a metabolite predicted by EMMF that has not been previously documented as part of the CHO cell metabolic model.
| S-EPMC7241244 | biostudies-literature
Project description:A study of DXP synthase has revealed flexibility in the acceptor substrate binding pocket for nonpolar substrates and has uncovered new details of the catalytic mechanism to show that pyruvate can act as both donor and acceptor substrate.
| S-EPMC2761658 | biostudies-literature