Project description:The reactive adenosine derivative, adenosine 5'-O-[S-(4-hydroxy-2,3-dioxobutyl)]-thiophosphate (AMPS-HDB), contains a dicarbonyl group linked to the purine nucleotide at a position equivalent to the pyrophosphate region of NAD+. AMPS-HDB was used as a chemical label towards Candida boidinii formate dehydrogenase (CbFDH). AMPS-HDB reacts covalently with CbFDH, leading to complete inactivation of the enzyme activity. The inactivation kinetics of CbFDH fit the Kitz and Wilson model for time-dependent, irreversible inhibition (KD = 0.66 ± 0.15 mM, first order maximum rate constant k3 = 0.198 ± 0.06 min-1). NAD+ and NADH protects CbFDH from inactivation by AMPS-HDB, showing the specificity of the reaction. Molecular modelling studies revealed Arg174 as a candidate residue able to be modified by the dicarbonyl group of AMPS-HDB. Arg174 is a strictly conserved residue among FDHs and is located at the Rossmann fold, the common mononucleotide-binding motif of dehydrogenases. Arg174 was replaced by Asn, using site-directed mutagenesis. The mutant enzyme CbFDHArg174Asn was showed to be resistant to inactivation by AMPS-HDB, confirming that the guanidinium group of Arg174 is the target for AMPS-HDB. The CbFDHArg174Asn mutant enzyme exhibited substantial reduced affinity for NAD+ and lower thermostability. The results of the study underline the pivotal and multifunctional role of Arg174 in catalysis, coenzyme binding and structural stability of CbFDH.
Project description:Using glucose oxidase (GOx) and α-Zr(IV) phosphate nanoplates (α-ZrP) as a model system, a generally applicable approach to control enzyme-solid interactions via chemical modification of amino acid side chains of the enzyme is demonstrated. Net charge on GOx was systematically tuned by appending different amounts of polyamine to the protein surface to produce chemically modified GOx(n), where n is the net charge on the enzyme after the modification and ranged from -62 to +95 electrostatic units in the system. The binding of GOx(n) with α-ZrP nanosheets was studied by isothermal titration calorimetry (ITC) as well as by surface plasmon resonance (SPR) spectroscopy. Pristine GOx showed no affinity for the α-ZrP nanosheets, but GOx(n) where n ≥ -20 showed binding affinities exceeding (2.1 ± 0.6) × 106 M-1, resulting from the charge modification of the enzyme. A plot of GOx(n) charge vs Gibbs free energy of binding (ΔG) for n = +20 to n = +65 indicated an overall increase in favorable interaction between GOx(n) and α-ZrP nanosheets. However, ΔG is less dependent on the net charge for n > +45, as evidenced by the decrease in the slope as charge increased further. All modified enzyme samples and enzyme/α-ZrP complexes retained a significant amount of folding structure (examined by circular dichroism) as well as enzymatic activities. Thus, strong control over enzyme-nanosheet interactions via modulating the net charge of enzymes may find potential applications in biosensing and biocatalysis.
Project description:Aromatic interactions are well-known players in molecular recognition but their catalytic role in biological systems is less documented. Here, we report that a conserved aromatic stacking interaction between dUTPase and its nucleotide substrate largely contributes to the stabilization of the associative type transition state of the nucleotide hydrolysis reaction. The effect of the aromatic stacking on catalysis is peculiar in that uracil, the aromatic moiety influenced by the aromatic interaction is relatively distant from the site of hydrolysis at the alpha-phosphate group. Using crystallographic, kinetics, optical spectroscopy and thermodynamics calculation approaches we delineate a possible mechanism by which rate acceleration is achieved through the remote π-π interaction. The abundance of similarly positioned aromatic interactions in various nucleotide hydrolyzing enzymes (e.g. most families of ATPases) raises the possibility of the reported phenomenon being a general component of the enzymatic catalysis of phosphate ester hydrolysis.
Project description:Each year, millions of people worldwide contract tuberculosis (TB), the deadliest infection. The spread of infections with drug-resistant strains of Mycobacterium tuberculosis (Mtb) that are refractory to treatment poses a major global challenge. A major cause of resistance to antitubercular drugs of last resort, aminoglycosides, is overexpression of the Eis (enhanced intracellular survival) enzyme of Mtb, which inactivates aminoglycosides by acetylating them. We showed previously that this inactivation of aminoglycosides could be overcome by our recently reported Eis inhibitors that are currently in development as potential aminoglycoside adjunctive therapeutics against drug-resistant TB. To interrogate the robustness of the Eis inhibitors, we investigated the enzymatic activity of Eis and its inhibition by Eis inhibitors from three different structural families for nine single-residue mutants of Eis, including those found in the clinic. Three engineered mutations of the substrate binding site, D26A, W36A, and F84A, abolished inhibitor binding while compromising Eis enzymatic activity 2- to 3-fold. All other Eis mutants, including clinically observed ones, were potently inhibited by at least one inhibitor. This study helps position us one step ahead of Mtb resistance to Eis inhibitors as they are being developed for TB therapy.
Project description:Saccharopine reductase catalyzes the reductive amination of l-α-aminoadipate-δ-semialdehyde with l-glutamate to give saccharopine. Two mechanisms have been proposed for the reductase, one that makes use of enzyme side chains as acid-base catalytic groups, and a second, in which the reaction is catalyzed by enzyme-bound reactants. Site-directed mutagenesis was used to change acid-base candidates in the active site of the reductase to eliminate their ionizable side chain. Thus, the D126A, C154S and Y99F and several double mutant enzymes were prepared. Kinetic parameters in the direction of glutamate formation exhibited modest decreases, inconsistent with the loss of an acid-base catalyst. The pH-rate profiles obtained with all mutant enzymes decrease at low and high pH, suggesting acid and base catalytic groups are still present in all enzymes. Solvent kinetic deuterium isotope effects are all larger than those observed for wild type enzyme, and approximately equal to one another, suggesting the slow step is the same as that of wild type enzyme, a conformational change to open the site and release products (in the direction of saccharopine formation). Overall, the acid-base chemistry is likely catalyzed by bound reactants, with the exception of deprotonation of the α-amine of glutamate, which likely requires an enzyme residue.
Project description:Lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) were immobilized on octyl agarose. Then, the biocatalysts were chemically modified using glutaraldehyde, trinitrobenzenesulfonic acid or ethylenediamine and carbodiimide, or physically coated with ionic polymers, such as polyethylenimine (PEI) and dextran sulfate. These produced alterations of the enzyme activities have, in most cases, negative effects with some substrates and positive with other ones (e.g., amination of immobilized TLL increases the activity versus p-nitro phenyl butyrate (p-NPB), reduces the activity with R-methyl mandate by half and maintains the activity with S-isomer). The modification with PEI increased the biocatalyst activity 8-fold versus R-methyl mandelate. Enzyme stability was also modified, usually showing an improvement (e.g., the modification of immobilized TLL with PEI or glutaraldehyde enabled to maintain more than 70% of the initial activity, while the unmodified enzyme maintained less than 50%). The immobilized enzymes were also mineralized by using phosphate metals (Zn2+, Co2+, Cu2+, Ni2+ or Mg2+), and this affected also the enzyme activity, specificity (e.g., immobilized TLL increased its activity after zinc mineralization versus triacetin, while decreased its activity versus all the other assayed substrates) and stability (e.g., the same modification increase the residual stability from almost 0 to more than 60%). Depending on the enzyme, a metal could be positively, neutrally or negatively affected for a specific feature. Finally, we analyzed if the chemical modification could, somehow, tune the effects of the mineralization. Effectively, the same mineralization could have very different effects on the same immobilized enzyme if it was previously submitted to different physicochemical modifications. The same mineralization could present different effects on the enzyme activity, specificity or stability, depending on the previous modification performed on the enzyme, showing that these previous enzyme modifications alter the effects of the mineralization on enzyme features. For example, TLL modified with glutaraldehyde and treated with zinc salts increased its activity using R-methyl mandelate, while almost maintaining its activity versus the other unaltered substrates, whereas the aminated TLL maintained its activity with both methyl mandelate isomers, while it decreased with p-NPB and triacetin. TLL was found to be easier to tune than CALB by the strategies used in this paper. In this way, the combination of chemical or physical modifications of enzymes before their mineralization increases the range of modification of features that the immobilized enzyme can experienced, enabling to enlarge the biocatalyst library.
Project description:Biomimicry valuably allows the understanding of the essential chemical components required to recapitulate biological function, yet direct strategies for evaluating the roles of amino acids in proteins can be limited by access to suitable, subtly-altered unnatural variants. Here we describe a strategy for dissecting the role of histidine residues in enzyme active sites using unprecedented, chemical, post-translational side-chain-β,γ C-N bond formation. Installation of dehydroalanine (as a "tag") allowed the testing of nitrogen conjugate nucleophiles in "aza-Michael"-1,4-additions (to "modify"). This allowed the creation of a regioisomer of His (iso-His, Hisiso) linked instead through its pros-Nπ atom rather than naturally linked via C4, as well as an aza-altered variant aza-Hisiso. The site-selective generation of these unnatural amino acids was successfully applied to probe the contributing roles (e.g., size, H-bonding) of His residues toward activity in the model enzymes subtilisin protease from Bacillus lentus and Mycobacterium tuberculosis pantothenate synthetase.
Project description:Bio-based polymer is considered as one of potentially renewable materials to reduce the consumption of petroleum resources. We report herein on the one-pot synthesis and development of unnatural-type bio-based polysaccharide, α-1,3-glucan. The synthesis can be achieved by in vitro enzymatic polymerization with GtfJ enzyme, one type of glucosyltransferase, cloned from Streptococcus salivarius ATCC 25975 utilizing sucrose, a renewable feedstock, as a glucose monomer source, via environmentally friendly one-pot water-based reaction. The structure of α-1,3-glucan is completely linear without branches with weight-average molecular weight (Mw) of 700 kDa. Furthermore, acetate and propionate esters of α-1,3-glucan were synthesized and characterized. Interestingly, α-1,3-glucan acetate showed a comparatively high melting temperature at 339 °C, higher than that of commercially available thermoplastics such as PET (265 °C) and Nylon 6 (220 °C). Thus, the discovery of crystalline α-1,3-glucan esters without branches with high thermal stability and melting temperature opens the gate for further researches in the application of thermoplastic materials.
Project description:Isopentenyl phosphate kinase (IPK) catalyzes the ATP-dependent phosphorylation of isopentenyl phosphate (IP) to form isopentenyl diphosphate (IPP) during biosynthesis of isoprenoid metabolites in Archaea. The structure of IPK from the archeaon Thermoplasma acidophilum (THA) was recently reported and guided the reconstruction of the IP binding site to accommodate the longer chain isoprenoid monophosphates geranyl phosphate (GP) and farnesyl phosphate (FP). We created four mutants of THA IPK with different combinations of alanine substitutions for Tyr70, Val73, Val130, and Ile140, amino acids with bulky side chains that limited the size of the side chain of the isoprenoid phosphate substrate that could be accommodated in the active site. The mutants had substantially increased GP kinase activity, with 20-200-fold increases in k(cat)(GP) and 30-130-fold increases in k(cat)(GP)/K(M)(GP) relative to those of wild-type THA IPK. The mutations also resulted in a 10(6)-fold decrease in k(cat)(IP)/K(M)(IP) compared to that of wild-type IPK. No significant change in the kinetic parameters for the cosubstrate ATP was observed, signifying that binding between the nucleotide binding site and the IP binding site was not cooperative. The shift in substrate selectivity from IP to GP, and to a lesser extent, FP, in the mutants could act as a starting point for the creation of more efficient GP or FP kinases whose products could be exploited for the chemoenzymatic synthesis of radiolabeled isoprenoid diphosphates.
Project description:We have recently shown that p-cyanophenylalanine (Cnf) and a thioamide can be used as a minimally perturbing Förster resonant energy transfer (FRET) pair to monitor protein conformation. We have also shown that thioamide analogues of natural amino acids can be incorporated into full-sized proteins through native chemical ligation. For intermolecular studies with Cnf/thioamide FRET pairs, Cnf can be incorporated into proteins expressed in Escherichia coli through unnatural amino acid mutagenesis using a Cnf-specific tRNA synthetase. For intramolecular studies, a Cnf-labeled protein fragment can be expressed in E. coli and then ligated to a thioamide-labeled peptide synthesized on solid phase. This combination of methods allows for rapid access to double-labeled proteins with a minimum of unnecessary chemical synthesis. We demonstrate the utility of this approach by studying the binding of peptides to the protein calmodulin and by determining the orientation of the N- and C-termini in the amyloidogenic protein α-synuclein.