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As part of ongoing efforts to design improved nerve agent antidotes, two X-ray crystal structures of Torpedo californica acetylcholinesterase ( TcAChE) bound to the bis-pyridinium oxime, Ortho-7, or its experimental bis-imidazolium analogue, 2BIM-7, were determined. Bis-oximes contain two oxime groups connected by a hydrophobic linker. One oxime group of Ortho-7 binds at the entrance to the active-site gorge near Trp279, and the second binds at the bottom near Trp84 and Phe330. In the Ortho-7- TcAChE complex the oxime at the bottom of the gorge was directed towards the nucleophilic Ser200. In contrast, the oxime group of 2BIM-7 was rotated away from Ser200 and the oxime at the entrance induced a significant conformational change in the peripheral anionic site (PAS) residue Trp279. The conformational change alters the surface of the PAS and positions the imidazolium oxime of 2BIM-7 further from Ser200. The relatively weaker binding and poorer reactivation of VX-inhibited, tabun-inhibited or sarin-inhibited human acetylcholinesterase by 2BIM-7 compared with Ortho-7 may in part be owing to the unproductively bound states caught in crystallo. Overall, the reactivation efficiency of 2BIM-7 was comparable to that of 2-pyridine aldoxime methyl chloride (2-PAM), but unlike 2-PAM the bis-imidazolium oxime lacks a fixed charge, which may affect its membrane permeability. [ABSTRACT FROM AUTHOR]

The metabolic versatility, tractability and rapid growth potential of the Vibrio spp. have made them increasingly attractive systems for investigating carbon cycling in the marine environment. In this study, an in silico subtractive proteomic strategy was used to identify a novel 101 kDa GH3 family β-glucosidase (LamN) that was found in bioluminescent Vibrio campbellii strains capable of utilizing the algal storage glucan laminarin. A heterologous overexpression system verified the sequence-predicted function of LamN as it enabled the growth of Escherichia coli on laminarin as a sole carbon source. Quantitative reverse transcription PCR analyses revealed that V. campbellii grown on laminarin demonstrated a 4- to 314-fold induction of lamN gene expression when compared to the same strains grown on glucose or glycerol. Corresponding tandem mass spectrometric analyses detected LamN protein expression only in cells grown on laminarin. Heterologous expression, purification and biochemical characterization identified LamN as a heat stable laminarinase with β-1,3, β-1,4 and β-1,6 glucosidase activity. Collectively, these data identify an enzyme that may allow V. campbellii to exploit some of the most abundant polysaccharides associated with deteriorating phytoplankton blooms and provide support for the potential involvement of V. campbellii in the formation of bioluminescent milky seas. [ABSTRACT FROM AUTHOR]

A study presents the results of previous kinetic stereochemical, structural and binding studies which investigated the catalytic role of three arginine residues, namely, Arg-11, Arg-39 and Arg-61 in 4-oxalocrotonate tautomerase. Findings revealed that Arg-11 is important in both substrate binding and catalysis. Arg-39 contributes largely to catalysis while Arg-61 plays little or no role in substrate binding and catalysis. Experimental procedures and results are presented.

Vitetta and colleagues identified and characterized a putative vascular leak peptide (VLP) consensus sequence in recombinant ricin toxin A-chain (RTA) that contributed to dose-limiting human toxicity when RTA was administered intravenously in large quantities during chemotherapy. We disrupted this potentially toxic site within the more stable RTA1-33/44-198 vaccine immunogen and determined the impact of these mutations on protein stability, structure and protective immunogenicity using an experimental intranasal ricin challenge model in BALB/c mice to determine if the mutations were compatible. Single amino acid substitutions at the positions corresponding with RTA D75 (to A, or N) and V76 (to I, or M) had minor effects on the apparent protein melting temperature of RTA1-33/44-198 but all four variants retained greater apparent stability than the parent RTA. Moreover, each VLP(-) variant tested provided protection comparable with that of RTA1-33/44-198 against supralethal intranasal ricin challenge as judged by animal survival and several biomarkers. To understand better how VLP substitutions and mutations near the VLP site impact epitope structure, we introduced a previously described thermal stabilizing disulfide bond (R48C/T77C) along with the D75N or V76I substitutions in RTA1-33/44-198. The D75N mutation was compatible with the adjacent stabilizing R48C/T77C disulfide bond and the Tm was unaffected, whereas the V76I mutation was less compatible with the adjacent disulfide bond involving C77. A crystal structure of the RTA1-33/44-198 R48C/T77C/D75N variant showed that the structural integrity of the immunogen was largely conserved and that a stable immunogen could be produced from E. coli. We conclude that it is feasible to disrupt the VLP site in RTA1-33/44-198 with little or no impact on apparent protein stability or protective efficacy in mice and such variants can be stabilized further by introduction of a disulfide bond. [ABSTRACT FROM AUTHOR]

γ-Glutamyl transpeptidase (GGT) is a two-substrate enzyme that plays a central role in glutathione metabolism and is a potential target for drug design. GGT catalyzes the cleavage of γ-glutamyl donor substrates and the transfer of the γ-glutamyl moiety to an amine of an acceptor substrate or water. Although structures of bacterial GGT have revealed details of the protein-ligand interactions at the donor site, the acceptor substrate site is relatively undefined. The recent identification of a species-specific acceptor site inhibitor, OU749, suggests that these inhibitors may be less toxic than glutamine analogues. Here we investigated the donor and acceptor substrate preferences of Bacillus anthracis GGT (CapD) and applied computational approaches in combination with kinetics to probe the structural basis of the enzyme's substrate and inhibitor binding specificities and compare them with human GGT. Site-directed mutagenesis studies showed that the R432A and R520S variants exhibited 6- and 95-fold decreases in hydrolase activity, respectively, and that their activity was not stimulated by the addition of the l-Cys acceptor substrate, suggesting an additional role in acceptor binding and/or catalysis of transpeptidation. Rat GGT (and presumably HuGGT) has strict stereospecificity for l-amino acid acceptor substrates, while CapD can utilize both l- and d-acceptor substrates comparably. Modeling and kinetic analysis suggest that R520 and R432 allow two alternate acceptor substrate binding modes for l- and d-acceptors. R432 is conserved in Francisella tularensis, Yersinia pestis, Burkholderia mallei, Helicobacter pylori and Escherichia coli, but not in human GGT. Docking and MD simulations point toward key residues that contribute to inhibitor and acceptor substrate binding, providing a guide to designing novel and specific GGT inhibitors. [ABSTRACT FROM AUTHOR]

Cathepsin A (CatA, EC 3.4.16.5, UniProtKB P10619 ) is a human lysosomal carboxypeptidase. Counterintuitively, crystal structures of CatA and its homologues show a cluster of Glu and Asp residues binding the C-terminal carboxylic acid of the product or inhibitor. Each of these enzymes functions in an acidic environment and contains a highly conserved pair of Glu residues with side chain carboxyl group oxygens that are approximately 2.3-2.6 Å apart. In small molecules, carboxyl groups separated by ∼3 Å can overcome the repulsive interaction by protonation of one of the two groups. The p K a of one group increases (p K a ∼ 11) and can be as much as ∼6 pH units higher than the paired group. Consequently, at low and neutral pH, one carboxylate can carry a net negative charge while the other can remain protonated and neutral. In CatA, E69 and E149 form a Glu pair that is important to catalysis as evidenced by the 56-fold decrease in k cat / K m in the E69Q/E149Q variant. Here, we have measured the pH dependencies of log( k cat ), log( K m ), and log( k cat / K m ) for wild type CatA and its variants and have compared the measured p K a with calculated values. We propose a substrate-assisted mechanism in which the high p K a of E149 (>8.5) favors the binding of the carboxylate form of the substrate and promotes the abstraction of the proton from H429 of the catalytic triad effectively decreasing its p K a in a low-pH environment. We also identify a similar motif consisting of a pair of histidines in S -formylglutathione hydrolase.

Inclusion of affinity tags has greatly facilitated process development for protein antigens, primarily for their recovery from complex mixtures. Although generally viewed as supportive of product development, affinity tags may have unintended consequences on protein solubility, susceptibility to aggregation, and immunogenicity. Merozoite surface protein 1 (MSP1), an erythrocytic stage protein of Plasmodium falciparum and a candidate malaria vaccine, was used to evaluate the impact of a metal ion affinity-tag on both protein structure and the induction of immunity. To this end, codon harmonized gene sequences from the P. falciparum MSP1(42) of FVO and 3D7 parasites were cloned and purified with and without a histidine (His) tag. We report on the influence of His-affinity tags on protein expression levels, solubility, secondary structure, thermal denaturation, aggregation and the impact on humoral and cellular immune responses in mice. While the overall immunogenicity induced by His-tagged MSP1(42) proteins is greater, the fine specificity of the humoral and cellular immune responses is altered relative to anti-parasitic antibody activity and the breadth of T-cell responses. Thus, the usefulness of protein tags may be outweighed by their potential impact on structure and function, stressing the need for caution in their use. See accompanying commentary by Randolph DOI: 10.1002/biot.201100459., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Over the past two decades, prokaryotic expression systems have been widely exploited for the bioproduction of many therapeutic proteins. Much of the success can be attributed to the implementation of basic principles of prokaryotic protein translation and protein folding to the problems of heterologous expression (e.g. codon usage substitutions, tRNA isoacceptor co-expression, chaperone co-expression); however, expression in a heterologous host still remains an empirical process. To improve heterologous protein expression further we have developed an algorithm termed "codon harmonization" that best approximates codon usage frequencies from the native host and adjusts these for use in the heterologous system. The success of this methodology may be due to improved protein folding during translation. Although so far exclusively applied to Escherichia coli, codon harmonization may provide a general strategy for improving the expression of soluble, functional proteins during heterologous host expression.