Design of surface-active artificial enzyme particles to stabilize Pickering emulsions for high-performance biphasic biocatalysis

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Surface-active artificial enzymes (SAEs) are designed and constructed by a general and novel strategy. These SAEs can simultaneously stabilize Pickering emulsions and catalyze biphasic biotransformation with superior enzymatic stability and good re-usability; for example, for the interfacial conversion of hydrophobic p-nitrophenyl butyrate into yellow water-soluble p-nitrophenolate catalyzed by esterase-mimic SAE

Mining electron density for functionally relevant protein polysterism in crystal structures.

This review focuses on conceptual and methodological advances in our understanding and characterization of the conformational heterogeneity of proteins. Focusing on X-ray crystallography, we describe how polysterism, the interconversion of pre-existing conformational substates, has traditionally been analyzed by comparing independent crystal structures or multiple chains within a single crystal asymmetric unit. In contrast, recent studies have focused on mining electron density maps to reveal previously 'hidden' minor conformational substates. Functional tests of the importance of minor states suggest that evolutionary selection shapes the entire conformational landscape, including uniquely configured conformational substates, the relative distribution of these substates, and the speed at which the protein can interconvert between them. An increased focus on polysterism may shape the way protein structure and function is studied in the coming years

Functional Trade-Offs in Promiscuous Enzymes Cannot Be Explained by Intrinsic Mutational Robustness of the Native Activity.

The extent to which an emerging new function trades off with the original function is a key characteristic of the dynamics of enzyme evolution. Various cases of laboratory evolution have unveiled a characteristic trend; a large increase in a new, promiscuous activity is often accompanied by only a mild reduction of the native, original activity. A model that associates weak trade-offs with "evolvability" was put forward, which proposed that enzymes possess mutational robustness in the native activity and plasticity in promiscuous activities. This would enable the acquisition of a new function without compromising the original one, reducing the benefit of early gene duplication and therefore the selection pressure thereon. Yet, to date, no experimental study has examined this hypothesis directly. Here, we investigate the causes of weak trade-offs by systematically characterizing adaptive mutations that occurred in two cases of evolutionary transitions in enzyme function: (1) from phosphotriesterase to arylesterase, and (2) from atrazine chlorohydrolase to melamine deaminase. Mutational analyses in various genetic backgrounds revealed that, in contrast to the prevailing model, the native activity is less robust to mutations than the promiscuous activity. For example, in phosphotriesterase, the deleterious effect of individual mutations on the native phosphotriesterase activity is much larger than their positive effect on the promiscuous arylesterase activity. Our observations suggest a revision of the established model: weak trade-offs are not caused by an intrinsic robustness of the native activity and plasticity of the promiscuous activity. We propose that upon strong adaptive pressure for the new activity without selection against the original one, selected mutations will lead to the largest possible increases in the new function, but whether and to what extent they decrease the old function is irrelevant, creating a bias towards initially weak trade-offs and the emergence of generalist enzymes.This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC, Discovery Grant RGPIN 418262- 12, http://www.nserc-crsng.gc.ca/), the Biotechnology and Biological Sciences Research Council (BBSRC, Grant BB/L002469/1, http://www. bbsrc.ac.uk/), the European Research Council (ERC, Advanced Investigator Grant 695669, https:// erc.europa.eu/), and the Human Frontiers Science Program (Grant RGP0006/2013, http://www.hfsp. org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Evolutionary expansion of the amidohydrolase superfamily in bacteria in response to the synthetic compounds molinate and diuron

The amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible

Archaeal Enzymes and Applications in Industrial Biocatalysts

Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in “extreme” conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches.University of ExeterWellcome TrustBBSRCEPSRCTechnology Strategy Board, UKEU Framework

Improving the 'tool box' for robust industrial enzymes.

This is the final version of the article. Available from Springer Verlag via the DOI in this record.The speed of sequencing of microbial genomes and metagenomes is providing an ever increasing resource for the identification of new robust biocatalysts with industrial applications for many different aspects of industrial biotechnology. Using 'natures catalysts' provides a sustainable approach to chemical synthesis of fine chemicals, general chemicals such as surfactants and new consumer-based materials such as biodegradable plastics. This provides a sustainable and 'green chemistry' route to chemical synthesis which generates no toxic waste and is environmentally friendly. In addition, enzymes can play important roles in other applications such as carbon dioxide capture, breakdown of food and other waste streams to provide a route to the concept of a 'circular economy' where nothing is wasted. The use of improved bioinformatic approaches and the development of new rapid enzyme activity screening methodology can provide an endless resource for new robust industrial biocatalysts.This mini-review will discuss several recent case studies where industrial enzymes of 'high priority' have been identified and characterised. It will highlight specific hydrolase enzymes and recent case studies which have been carried out within our group in Exeter.The author would like to thank the University of Exeter, the Wellcome Trust, the BBSRC, EPSRC and Technology Strategy Board, UK, for sponsoring research in the JAL group at the Exeter Biocatalysis Centre. The BBSRC funded ERA-IB grant BB/L002035/1 and the University of Exeter are thanked for support. Thanks are also given to the EU Framework 7 grant ‘HOTZYME’ entitled Systematic Screening of Organisms from Hot Environments, Grant Number 265933

OpdA, a bacterial organophosphorus hydrolase, prevents lethality in rats after poisoning with highly toxic organophosphorus pesticides

Organophosphorus (OP) pesticides poison more than 3,000,000 people every year in the developing world, mostly through intentional self-poisoning. Advances in medical therapy for OP poisoning have lagged, and current treatment is not highly effective with mortality of up to 40% in even the most advanced Western medical facilities. Administration of a broadly active bacterial OP hydrolase to patients in order to hydrolyze OPs in circulation might allow current therapies to be more effective. The objective of this work was to evaluate the efficacy of a new recombinant bacterial OP hydrolase (OpdA), cloned from Agrobacterium radiobacter, in rat models of two chemically distinct but highly toxic and rapidly acting OP pesticides: dichlorvos and parathion. Without OpdA treatment, median time to death in rats poisoned with 3x LD(50) of dichlorvos or parathion was 6 min and 25.5 min, respectively. Administration of a single dose of OpdA immediately after dichlorvos resulted in 100% survival at 24h, with no additional antidotal therapy. After parathion poisoning, OpdA alone caused only a delay to death. However, an additional two doses of OpdA resulted in 62.5% survival at 24 h after parathion poisoning. In combination with pralidoxime therapy, a single dose of OpdA increased survival to 75% after parathion poisoning. Our results demonstrate that OpdA is able to improve survival after poisoning by two chemically distinct and highly toxic OP pesticides