Nectrisine Biosynthesis Genes in Thelonectria discophora SANK 18292: Identification and Functional Analysis.

The fungus Thelonectria discophora SANK 18292 produces the iminosugar nectrisine, which has a nitrogen-containing heterocyclic 5-membered ring and acts as a glycosidase inhibitor. In our previous study, an oxidase (designated NecC) that converts 4-amino-4-deoxyarabinitol to nectrisine was purified from T. discophora cultures. However, the genes required for nectrisine biosynthesis remained unclear. In this study, the nectrisine biosynthetic gene cluster in T. discophora was identified from the contiguous genome sequence around the necC gene. Gene disruption and complementation studies and heterologous expression of the gene showed that necA, necB, and necC could be involved in nectrisine biosynthesis, during which amination, dephosphorylation, and oxidation occur. It was also demonstrated that nectrisine could be produced by recombinant Escherichia coli coexpressing the necA, necB, and necC genes. These findings provide the foundation to develop a bacterial production system for nectrisine or its intermediates through genetic engineering. IMPORTANCE: Iminosugars might have great therapeutic potential for treatment of many diseases. However, information on the genes for their biosynthesis is limited. In this study, we report the identification of genes required for biosynthesis of the iminosugar nectrisine in Thelonectria discophora SANK 18292, which was verified by disruption, complementation, and heterologous expression of the genes involved. We also demonstrate heterologous production of nectrisine by recombinant E. coli, toward developing an efficient production system for nectrisine or its intermediates through genetic engineering.

Evaluation of fluoropyruvate as nucleophile in reactions catalysed by N-acetyl neuraminic acid lyase variants: scope, limitations and stereoselectivity.

The catalysis of reactions involving fluoropyruvate as donor by N-acetyl neuraminic acid lyase (NAL) variants was investigated. Under kinetic control, the wild-type enzyme catalysed the reaction between fluoropyruvate and N-acetyl mannosamine to give a 90 : 10 ratio of the (3R,4R)- and (3S,4R)-configured products; after extended reaction times, equilibration occurred to give a 30 : 70 mixture of these products. The efficiency and stereoselectivity of reactions of a range of substrates catalysed by the E192N, E192N/T167V/S208V and E192N/T167G NAL variants were also studied. Using fluoropyruvate and (2R,3S)- or (2S,3R)-2,3-dihydroxy-4-oxo-N,N-dipropylbutanamide as substrates, it was possible to obtain three of the four possible diastereomeric products; for each product, the ratio of anomeric and pyranose/furanose forms was determined. The crystal structure of S. aureus NAL in complex with fluoropyruvate was determined, assisting rationalisation of the stereochemical outcome of C-C bond formation.

Biosynthesis of nectrisine in Thelonectria discophora SANK 18292.

Nectrisine, an iminosugar with a heterocyclic nitrogen-containing 5-membered ring, acts as a glycosidase inhibitor. Thelonectria discophora SANK 18292, a fungus, was identified as a nectrisine producer from its microbial library in our screening for nectrisine producing microorganisms. Biosynthesis of nectrisine produced by the fungus was studied using stable isotope tracer techniques. Incorporation of (13)C-labeled d-ribose and d-xylose into nectrisine was confirmed by mass spectrometry and (13)C NMR spectroscopy, which suggested that these were its precursors. Chromatographic separation of the hot water extract from the culture broth afforded not only nectrisine, but also substantial amounts of 4-amino-4-deoxyarabinitol. Incubation of the latter with the crude enzyme of the fungus at room temp. caused an increase in levels of nectrisine together with a decrease in amounts of the administered potential precursor suggesting that it is a biosynthetic intermediate. From these results, a biosynthetic pathway to nectrisine is proposed via d-xylulose 5-phosphate and 4-amino-4-deoxyarabinitol by the pentose phosphate pathway.

Chemo-enzymatic synthesis and glycosidase inhibitory properties of DAB and LAB derivatives.

A chemo-enzymatic strategy for the preparation of 2-aminomethyl derivatives of (2R,3R,4R)-2-(hydroxymethyl)pyrrolidine-3,4-diol (also called 1,4-dideoxy-1,4-imino-D-arabinitol, DAB) and its enantiomer LAB is presented. The synthesis is based on the enzymatic preparation of DAB and LAB followed by the chemical modification of their hydroxymethyl functionality to afford diverse 2-aminomethyl derivatives. This strategy leads to novel aromatic, aminoalcohol and 2-oxopiperazine DAB and LAB derivatives. The compounds were preliminarily explored as inhibitors of a panel of commercial glycosidases, rat intestinal disaccharidases and against Mycobacterium tuberculosis, the causative agent of tuberculosis. It was found that the inhibitory profile of the new products differed considerably from the parent DAB and LAB. Furthermore, some of them were active inhibiting the growth of M. tuberculosis.

Energy landscapes associated with macromolecular conformational changes from endpoint structures.

Conformational changes modulate macromolecular function by promoting the specific binding of ligands (such as in antigen recognition) or the stabilization of transition states in enzymatic reactions. However, quantitative characterization of the energetics underlying dynamic structural interconversions is still challenging and lacks a unified method. Here, we introduce a novel in silico approach based on the combined use of essential dynamics sampling and nonequilibrium free-energy calculations to obtain quantitative data on conformational energy landscapes. This technique allows the unbiased investigation of highly complex rearrangements, and does not require the crucial definition of user-defined collective variables. We show that free-energy values derived from profiles connecting the unliganded and ligand-bound X-ray structures of a bacterial nucleoside hydrolase match the experimental binding constant. This approach also provides first evidence for a rate-limiting character of the conformational transition in this enzyme, and an unexpected role of the protonation state of a single residue in regulating substrate binding and product release.

Active site plasticity revealed from the structure of the enterobacterial N-ribohydrolase RihA bound to a competitive inhibitor.

BACKGROUND: Pyrimidine-preferring N-ribohydrolases (CU-NHs) are a class of Ca2+-dependent enzymes that catalyze the hydrolytic cleavage of the N-glycosidic bond in pyrimidine nucleosides. With the exception of few selected organisms, their physiological relevance in prokaryotes and eukaryotes is yet under investigation. RESULTS: Here, we report the first crystal structure of a CU-NH bound to a competitive inhibitor, the complex between the Escherichia coli enzyme RihA bound to 3, 4-diaminophenyl-iminoribitol (DAPIR) to a resolution of 2.1 A. The ligand can bind at the active site in two distinct orientations, and the stabilization of two flexible active site regions is pivotal to establish the interactions required for substrate discrimination and catalysis. CONCLUSIONS: A comparison with the product-bound RihA structure allows a rationalization of the structural rearrangements required for an enzymatic catalytic cycle, highlighting a substrate-assisted cooperative motion, and suggesting a yet overlooked role of the conserved His82 residue in modulating product release. Differences in the structural features of the active sites in the two homologous CU-NHs RihA and RihB from E. coli provide a rationale for their fine differences in substrate specificity. These new findings hint at a possible role of CU-NHs in the breakdown of modified nucleosides derived from RNA molecules.

Both thiamine uptake and biosynthesis of thiamine precursors are required for intracellular replication of Listeria monocytogenes.

Thiamine pyrophosphate is an essential cofactor involved in central metabolism and amino acid biosynthesis and is derived from thiamine (vitamin B(1)). The extent to which this metabolite is available to bacterial pathogens replicating within host cells is still little understood. Growth studies using modified minimal Welshimer's broth (mMWB) supplemented with thiamine or the thiamine precursor hydroxymethylpyrimidine (HMP) showed that Listeria monocytogenes, in agreement with bioinformatic prediction, is able to synthesize thiamine only in the presence of HMP. This appears to be due to a lack of ThiC, which is involved in HMP synthesis. The knockout of thiD (lmo0317), which probably catalyzes the phosphorylation of HMP, inhibited growth in mMWB supplemented with HMP and reduced the replication rate of L. monocytogenes in epithelial cells. Mutation of a predicted thiamine transporter gene, lmo1429, led to reduced proliferation of L. monocytogenes in mMWB containing thiamine or thiamine phosphates and also within epithelial cells but had no influence on the expression of the virulence factors Hly and ActA. The toxic thiamine analogue pyrithiamine inhibited growth of wild-type strain EGD but not of the transporter mutant EGDDeltathiT. We also demonstrated that ThiT binds thiamine, a finding compatible with ThiT acting as the substrate-binding component of a multimeric thiamine transporter complex. These data provide experimental evidence that Lmo1429 homologs including Bacillus YuaJ are necessary for thiamine transport in gram-positive bacteria and are therefore proposed to be annotated "ThiT." Taken together, these data indicate that concurrent thiamine uptake and biosynthesis of thiamine precursors is a strategy of L. monocytogenes and possibly other facultative intracellular pathogens to enable proliferation within the cytoplasm.