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To search for a novel thermostable esterase for optimized industrial applications, esterase from a thermophilic eubacterium species, Thermoanaerobacter tengcongensis MB4, was purified and characterized in this work. Sequence analysis of T. tengcongensis esterase with other homologous esterases of the same family revealed an apparent tail at the C-terminal that is not conserved across the esterase family. Hence, it was hypothesized that the tail is unlikely to have an essential structural or catalytic role. However, there is no documented report of any role for this tail region. We probed the role of the C-terminal domain on the catalytic activity and substrate preference of T. tengcongensis esterase EstA3 with a view to see how it could be engineered for enhanced properties. To achieve this, we cloned, expressed, and purified the wild-type and the truncated versions of the enzyme. In addition, a naturally occurring member of the family (from Brevibacillus brevis) that lacks the C-terminal tail was also made. In vitro characterization of the purified enzymes showed that the C-terminal domain contributes significantly to the catalytic activity and distinct substrate preference of T. tengcongensis esterase EstA3. All three recombinant enzymes showed the highest preference for paranitrophenyl butyrate (pNPC4), which suggests they are true esterases, not lipases. Kinetic data revealed that truncation had a slight effect on the substrate-binding affinity. Thus, the drop in preference towards long-chain substrates might not be a result of substrate binding affinity alone. The findings from this work could form the basis for future protein engineering allowing the modification of esterase catalytic properties through domain swapping or by attaching a modular protein domain. [ABSTRACT FROM AUTHOR]

The mechanism of modulation of vanadate inhibition of alkaline phosphatase activity by catalytic cofactors has not been fully characterized. We investigated the effect of the interaction of catalytic cofactors (Mg2+ and Zn2+) and vanadate (an active site inhibitor) on the rate of hydrolysis of para-nitrophenyl phosphate (pNPP) (monoesterase reaction) by calf intestinal alkaline phosphatase (CIAP). The results showed that vanadate significantly inhibited' cofactor-free' CIAP, and the inhibition was relieved by the presence of the catalytic cofactors in the reaction. Our results show that the absence of the cofactors did not significantly alter the Km of the reaction, but caused a decrease in the Vmax. Kinetic analyses showed that vanadate inhibited CIAP-catalyzed hydrolysis of pNPP by decreasing the Vmax and increasing the KVm of the reaction. The presence of cofactors in the reaction alleviated the effect of vanadate by increasing the Vmax and decreasing the Km. The activity of the dialyzed CIAP was increased by the addition of catalytic cofactors to vanadate-inhibited enzyme. This study provides preliminary data that reversible inhibition of CIAP is subject to the influence of catalytic cofactors. Further studies will reveal detailed mechanistic aspects of this observation and its significance in the biological system. [ABSTRACT FROM AUTHOR]

G-quadruplex DNAzymes are short DNA aptamers with repeating G4 quartets bound in a non-covalent complex with hemin. These G4/Hemin structures exhibit versatile peroxidase-like catalytic activity with a wide range of potential applications in biosensing and biotechnology. Current efforts are aimed at gaining a better understanding of the molecular mechanism of DNAzyme catalysis as well as devising strategies for improving their catalytic efficiency. Multimerisation of discrete units of G-quadruplexes to form multivalent DNAzyes is an emerging design strategy aimed at enhancing the peroxidase activities of DNAzymes. While this approach holds promise of generating more active multivalent G-quadruplex DNAzymes, few examples have been studied and it is not clear what factors determine the enhancement of catalytic activities of multimeric DNAzymes. In this study, we report the design and characterisation of multimers of five G-quadruplex sequences (AS1411, Bcl-2, c-MYC, PS5.M and PS2.M). Our results show that multimerisation of G-quadruplexes that form parallel structure (AS1411, Bcl-2, c-MYC) leads to significant rate enhancements characteristic of cooperative and/or synergistic interactions between the monomeric units. In contrast, multimerisation of DNA sequences that form non-parallel structures (PS5.M and PS2.M) did not exhibit similar levels of synergistic increase in activities. These results show that design of multivalent G4/Hemin structures could lead to a new set of versatile and efficient DNAzymes with enhanced capacity to catalyse peroxidase-mimic reactions. [ABSTRACT FROM AUTHOR]