OleD Loki as a Catalyst for Tertiary Amine and Hydroxamate Glycosylation

We describe the ability of an engineered glycosyltransferase (OleD Loki) to catalyze the N‐glycosylation of tertiary‐amine‐containing drugs and trichostatin hydroxamate glycosyl ester formation. As such, this study highlights the first bacterial model catalyst for tertiary‐amine N‐glycosylation and further expands the substrate scope and synthetic potential of engineered OleDs. In addition, this work could open the door to the discovery of similar capabilities among other permissive bacterial glycosyltransferases

Structure and Specificity of a Permissive Bacterial C-Prenyltransferase

This study highlights the biochemical and structural characterization of the L-tryptophan C6 C-prenyltransferase (C-PT) PriB from Streptomyces sp. RM-5-8. PriB was found to be uniquely permissive to a diverse array of prenyl donors and acceptors including daptomycin. Two additional PTs also produced novel prenylated daptomycins with improved antibacterial activities over the parent drug

Structure and Specificity of a Permissive Bacterial C-prenyltransferase

This study highlights the biochemical and structural characterization of the L-tryptophan C6 C-prenyltransferase (C-PT) PriB from Streptomyces sp. RM-5-8. PriB was found to be uniquely permissive to a diverse array of prenyl donors and acceptors including daptomycin. Two additional PTs also produced novel prenylated daptomycins with improved antibacterial activities over the parent drug

Structural Dynamics of a Methionine γ-lyase for Calicheamicin Biosynthesis: Rotation of the Conserved Tyrosine Stacking with Pyridoxal Phosphate

CalE6 from Micromonospora echinospora is a (pyridoxal 5′ phosphate) PLP-dependent methionine γ-lyase involved in the biosynthesis of calicheamicins. We report the crystal structure of a CalE6 2-(N-morpholino)ethanesulfonic acidcomplex showing ligand-induced rotation of Tyr100, which stacks with PLP, resembling the corresponding tyrosine rotation of true catalytic intermediates of CalE6 homologs. Elastic network modeling and crystallographic ensemble refinement reveal mobility of the N-terminal loop, which involves both tetrameric assembly and PLP binding. Modeling and comparative structuralanalysis of PLP-dependent enzymes involved in Cys/Met metabolism shine light on the functional implications of the intrinsic dynamic properties of CalE6 in catalysis and holoenzyme maturation

Crystallographic, Kinetic and Computational Studies on the Reaction Mechanism of Xanthine Oxidoreductase

Xanthine oxidoreductase is a molybdenum-containing enzyme which catalyzes the hydroxylation on sp2 hybridized carbon centers of a broad family of substrates including purines, aldehydes and various other heterocycles. It catalyzes the sequential hydroxylation of physiological substrate hypoxanthine to uric acid. Deposition of uric acid crystals in human joints with accompanying inflammation is the major cause of gout. The production of reactive oxygen species by xanthine oxidase is implicated in the pathology of various inflammatory and cardiovascular diseases. The current study mainly involves: (1) X-ray crystallography to elucidate the orientations of various substrates at the active site of bovine xanthine oxidase. Our observation of a single dominant productive orientation of xanthine, alternative orientations of hypoxanthine, a single nonproductive orientation of guanine and the dominant nonproductive orientation of indole-3-aldehyde correlates well with different catalytic activities of xanthine oxidase with these substrates and suggests the existence of dynamic sampling of substrate orientations at enzyme active site. (2) X-ray crystallography to reveal the orientations of inhibitors arsenite and quercetin at the active site of xanthine oxidase. The binding modes of these inhibitors provide structural basis for the mechanism of inhibition and insights into inhibitor design for potential therapeutics. (3) UV-visible spectroscopy to quantitatively characterize the kinetics and catalytic specificity in the reactions of sequential hydroxylation of hypoxanthine to uric acid. We conclude that the hydroxylation of hypoxanthine by xanthine oxidase is strictly specific toward C-2 over C-8, although 6,8-dihydroxypurine is an effective substrate as xanthine both of which can be converted to uric acid by xanthine oxidase. (4) Primary deuterium kinetic isotopic effect (KIE) study on the wild-type enzyme and Gln197 mutants of R. capsulatus xanthine dehydrogenase. The small apparent KIE on kcat suggests that hydrogen transfer step is neither rate-limiting for the wild-type enzyme nor for the mutants. We identify gain-of-function Q197A and loss-of-function Q197E mutants. (5) Computation of pKa of Glu802/232 of xanthine oxidoreductase and kinetic characterization of E232Q mutant. Our data suggest that Glu802/232 has an acidic pKa upon binding of xanthine or hypoxanthine which supports its catalytic role in facilitating proton tautomerization

Government Microblog Information Exchange Efficiency and Its Influencing Factors for Emergency Management

[Purpose/Significance] This paper aimed at evaluating information exchange efficiency of fire-fighting microblogs, in order to guiding information exchange and promoting the information exchange efficiency in emergency management. There are two primary innovations in this paper. First, compared with the existing research on emergency management and DEA methods, the existing research on the application of government microblogs in emergency management mainly focused on qualitative research. The research on DEA method focused on the performance evaluation of enterprises and banks. This paper used the DEA method to quantitatively analyze the efficiency of information exchange in emergency management. Second, this paper used the DEA method to divide the efficiency of fire-fighting microblogs into two parts: DEA effective and non-DEA effective, and comprehensively explored measures to improve efficiency from these two parts. [Method/Process] This paper took the highest level of fire-fighting microblogs officially certified by the provinces, autonomous regions and municipalities as the research resources. In order to do this research, an indicator system based on current influence of government microblog and the efficiency of communication was constructed; DEA-BBC model was used to measure the efficiency of fire-fighting microblog information exchange; the micro and macro influencing factors of fire-fighting microblog information exchange efficiency were identified; finally some measures were put forward to improve the efficiency of information exchange on fire-fighting microblogging. DEA is a non-parametric test method commonly used for enterprise performance and efficiency evaluation in management science. It can be used to evaluate decision-making units of multiple input indicators and multiple output indicators. Beyond that, the decision-making results of the method are not affected by the dimensions of input indicators and output indicators. This method also does not need to make weight assumptions, so it can provide more objective analysis results to make contribution to refining more effective improvement measures. [Results/Conclusions] The information exchange efficiency of the fire-fighting microblogs was generally rated as the medium level. It is better for us to arrange various inputs of the microblog account reasonably, and make full use of resources, which could improve the efficiency of information exchange. In addition, the operation of fire-fighting microblogs in different regions should be based on regional characteristics and period characteristics; local governments ought to strengthen network infrastructure construction and resource investment to eliminate the "digital divide"; government agencies should improve management systems, standardize the words and deeds of netizens and the daily work of relevant personnel. Microblog plays an important role in emergency management, which is also a necessary condition for the vigorous development of China's Internet governance

Time-resolved x-ray crystallography capture of a slow reaction tetrahydrofolate intermediate

Time-resolved crystallography is a powerful technique to elucidate molecular mechanisms at both spatial (angstroms) and temporal (picoseconds to seconds) resolutions. We recently discovered an unusually slow reaction at room temperature that occurs on the order of days: the in crystalline reverse oxidative decay of the chemically labile (6S)-5,6,7,8-tetrahydrofolate in complex with its producing enzyme Escherichia coli dihydrofolate reductase. Here, we report the critical analysis of a representative dataset at an intermediate reaction time point. A quinonoid-like intermediate state lying between tetrahydrofolate and dihydrofolate features a near coplanar geometry of the bicyclic pterin moiety, and a tetrahedral sp3 C6 geometry is proposed based on the apparent mFo-DFc omit electron densities of the ligand. The presence of this intermediate is strongly supported by Bayesian difference refinement. Isomorphous Fo-Fo difference map and multi-state refinement analyses suggest the presence of end-state ligand populations as well, although the putative intermediate state is likely the most populated. A similar quinonoid intermediate previously proposed to transiently exist during the oxidation of tetrahydrofolate was confirmed by polarography and UV-vis spectroscopy to be relatively stable in the oxidation of its close analog tetrahydropterin. We postulate that the constraints on the ligand imposed by the interactions with the protein environment might be the origin of the slow reaction observed by time-resolved crystallography

Substrate orientation and the origin of catalytic power in xanthine oxidoreductase

355-362With the chemical course of the reaction catalyzed by the molybdenum-containing hydroxylase xanthine oxidoreductase now relatively well-understood, efforts in the field have now turned to understanding the catalytic power of the enzyme in the context of its structure. The present minireview is an account of recent efforts, from the authors’ laboratory and elsewhere, towards understanding the role of active site amino acid residues in accelerating reaction rate. On the basis of recent site-directed mutagenesis work, in conjunction with protein X-ray crystallography, it is now possible to attribute the specific extent to which each contributes to transition state stabilization and the means by which this occurs