Engineering the Substrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display

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Highlights? Yeast surface display to engineer substrate recognition of adenylation (A) domains ? Synthesis of bisubstrate inhibitors for yeast selection of A domain library ? Identifying DhbE mutants specific for 3-hydroxybenzoic acid and 2-aminobenzoic acid ? Key residues in A domain for substrate recognition and transfer     

Substrate recognition and channeling of monomodules from the pikromycin polyketide synthase

The unique ability of the pikromycin (Pik) polyketide synthase to generate 12- and 14-membered ring macrolactones presents an opportunity to explore the fundamental processes underlying polyketide synthesis, specifically the mechanistic details of the chain extension process. We have overexpressed and purified PikAIII (module 5) and PikAIV (module 6) and assessed the ability of these proteins to generate tri- and tetraketide lactone products using N-acetylcysteamine-activated diketides and (14)C-methylmalonyl-CoA as substrates. Comparison of the stereochemical specificities for PikAIII and PikAIV and the reported values for the DEBS modules reveals significant differences between these systems.     

Mechanism-based inactivation by aromatization of the transaminase BioA involved in biotin biosynthesis in Mycobaterium tuberculosis

BioA catalyzes the second step of biotin biosynthesis, and this enzyme represents a potential target to develop new antitubercular agents. Herein we report the design, synthesis, and biochemical characterization of a mechanism-based inhibitor (1) featuring a 3,6-dihydropyrid-2-one heterocycle that covalently modifies the pyridoxal 5'-phosphate (PLP) cofactor of BioA through aromatization. The structure of the PLP adduct was confirmed by MS/MS and X-ray crystallography at 1.94 ? resolution. Inactivation of BioA by 1 was time- and concentration-dependent and protected by substrate. We used a conditional knock-down mutant of M. tuberculosis to demonstrate the antitubercular activity of 1 correlated with BioA expression, and these results provide support for the designed mechanism of action.     

Parameterization and Application of the General Amber Force Field to Model Fluoro Substituted Furanose Moieties and Nucleosides

Molecular mechanics force field calculations have historically shown significant limitations in modeling the energetic and conformational interconversions of highly substituted furanose rings. This is primarily due to the gauche effect that is not easily captured using pairwise energy potentials. In this study, we present a refinement to the set of torsional parameters in the General Amber Force Field (gaff) used to calculate the potential energy of mono, di-, and gem-fluorinated nucleosides. The parameters were optimized to reproduce the pseudorotation phase angle and relative energies of a diverse set of mono- and difluoro substituted furanose ring systems using quantum mechanics umbrella sampling techniques available in the IpolQ engine in the Amber suite of programs. The parameters were developed to be internally consistent with the gaff force field and the TIP3P water model. The new set of angle and dihedral parameters and partial charges were validated by comparing the calculated phase angle probability to those obtained from experimental nuclear magnetic resonance experiments.     

Binding energy of a Mott-Wannier exciton in a polarizable medium

A new effective interaction potential between an electron and a hole of a Mott-Wannier exciton in a polarizable medium is derived following a procedure similar to that of Haken. The variational polaron wave functions used are those first proposed by Haga. Using this effective interaction potential the values of the binding energy of an exciton are calculated variationally in several polar crystals. The values thus obtained agree very well with those derived from experimental measurements and are always considerably smaller than those calculated using Haken's potential.     

Stereochemistry of 1,2-elimination reactions at the E2-E1cB interface--tert-butyl 3-tosyloxybutanoate and its thioester

Experimental data on the stereoselectivity of base-catalyzed 1,2-elimination reactions that produce conjugated carbonyl compounds are scarce in spite of the importance of these reactions in organic and biochemistry. As part of a comprehensive study in this area, we have synthesized stereospecifically-deuterated beta-tosyloxybutanoate esters and thioesters and studied the stereoselectivity of their elimination reactions under non-ion pairing conditions. With the availability of both the (2R*,3R*) and (2R*,3S*) diastereomers the innate stereoselectivity could be determined unambiguously. (1)H and (2)H NMR data show that these substrates produce 5-6% syn elimination, the usual amount for acyclic substrates undergoing E2 reactions. Contrary to earlier suggestions, activation by a carbonyl group has virtually no influence upon the stereoselectivity. Elimination of the (2R*,3R*) diastereomer of the beta-tosyloxyester and thioester produces 21-25% of the (Z)-alkene, much more than observed with a poorer beta-nucleofuge. A relatively large amount of (Z)-alkene product seems to be a good marker for an E2 pathway, in which the transition state is E1cB-like, rather than an E1cB(irrev) mechanism. Syn KIE values were higher than those for anti elimination for the esters as well as the thioesters. Experimental challenges to the synthesis of stereospecifically-deuterated beta-tosyloxyesters are discussed.     

The cross-entropy method in multi-objective optimisation: An assessment

Solving multi-objective problems requires the evaluation of two or more conflicting objective functions, which often demands a high amount of computational power. This demand increases rapidly when estimating values for objective functions of dynamic, stochastic problems, since a number of observations are needed for each evaluation set, of which there could be many. Computer simulation applications of real-world optimisations often suffer due to this phenomenon. Evolutionary algorithms are often applied to multi-objective problems. In this article, the cross-entropy method is proposed as an alternative, since it has been proven to converge quickly in the case of single-objective optimisation problems. We adapted the basic cross-entropy method for multi-objective optimisation and applied the proposed algorithm to known test problems. This was followed by an application to a dynamic, stochastic problem where a computer simulation model provides the objective function set. The results show that acceptable results can be obtained while doing relatively few evaluations.     

Bisubstrate adenylation inhibitors of biotin protein ligase from Mycobacterium tuberculosis

The mycobacterial biotin protein ligase (MtBPL) globally regulates lipid metabolism in Mtb through the posttranslational biotinylation of acyl coenzyme A carboxylases involved in lipid biosynthesis that catalyze the first step in fatty acid biosynthesis and pyruvate coenzyme A carboxylase, a gluconeogenic enzyme vital for lipid catabolism. Here we describe the design, development, and evaluation of a rationally designed bisubstrate inhibitor of MtBPL. This inhibitor displays potent subnanomolar enzyme inhibition and antitubercular activity against multidrug resistant and extensively drug resistant Mtb strains. We show that the inhibitor decreases in?vivo protein biotinylation of key enzymes involved in fatty?acid biosynthesis and that the antibacterial activity is MtBPL dependent. Additionally, the gene encoding BPL was found to be essential in M.?smegmatis. Finally, the X-ray cocrystal structure of inhibitor bound MtBPL was solved providing detailed insight for further structure-activity analysis. Collectively, these data suggest that MtBPL is a promising target for further antitubercular therapeutic development.