Evolving a lipase for hydrolysis of natural triglycerides along with enhanced tolerance towards a protease and surfactants.

In the accompanying paper, we described evolving a lipase to the point where variants were soluble, stable and capable of degrading C8 TAG and C8 esters. These variants were tested for their ability to survive in an environment that might be encountered in a washing machine. Unfortunately, they were inactivated both by treatment with a protease used in laundry detergents and by very low concentrations of sodium dodecyl sulfate (SDS).

Improving on nature's shortcomings: evolving a lipase for increased lipolytic activity, expression and thermostability.

An enzyme must be soluble, stable, active and easy to produce to be useful in industrial applications. Not all enzymes possess these attributes. We set out to determine how many changes are required to convert an enzyme with poor properties into one that has useful properties.

Kinetics of Photocatalyzed Reactions: Five Lessons Learned.

Elucidation of kinetics of photocatalyzed chemical mechanisms occurring at interfaces (gas-solid, liquid-solid) has been challenging. We summarize here five lessons learned over five decades. 1.

High resolution crystal structure of a fluoride-inhibited organophosphate-degrading metallohydrolase.

Metal ion-dependent, organophosphate-degrading enzymes (OP hydrolases) have received increasing attention due to their ability to degrade and thus detoxify commonly used pesticides and nerve agents such as sarin and VX. These enzymes thus garner strong potential as bioremediators. The OP hydrolase from Agrobacterium radiobacter (OpdA) is one of the most efficient members of this group of enzymes.

Insights into an evolutionary strategy leading to antibiotic resistance.

Metallo-β-lactamases (MBLs) with activity towards a broad-spectrum of β-lactam antibiotics have become a major threat to public health, not least due to their ability to rapidly adapt their substrate preference. In this study, the capability of the MBL AIM-1 to evade antibiotic pressure by introducing specific mutations was probed by two alternative methods, i.e.

AIM-1: An Antibiotic-Degrading Metallohydrolase That Displays Mechanistic Flexibility.

Antibiotic resistance has emerged as a major threat to global health care. This is largely due to the fact that many pathogens have developed strategies to acquire resistance to antibiotics. Metallo-β-lactamases (MBL) have evolved to inactivate most of the commonly used β-lactam antibiotics.

Ca(II) Binding Regulates and Dominates the Reactivity of a Transition-Metal-Ion-Dependent Diesterase from Mycobacterium tuberculosis.

The diesterase Rv0805 from Mycobacterium tuberculosis is a dinuclear metallohydrolase that plays an important role in signal transduction by controlling the intracellular levels of cyclic nucleotides. As Rv0805 is essential for mycobacterial growth it is a promising new target for the development of chemotherapeutics to treat tuberculosis. The in vivo metal-ion composition of Rv0805 is subject to debate.

Directed Evolution of Enzymes for Industrial Biocatalysis.

Enzymes have the potential to catalyse a wide variety of chemical reactions. They are increasingly being sought as environmentally friendly and cost-effective alternatives to conventional catalysts used in industries ranging from bioremediation to applications in medicine and pharmaceutics. Despite the benefits, they are not without their limitations.

Altering the substrate specificity of methyl parathion hydrolase with directed evolution.

Many organophosphates (OPs) are used as pesticides in agriculture. They pose a severe health hazard due to their inhibitory effect on acetylcholinesterase. Therefore, detoxification of water and soil contaminated by OPs is important.

Pseudomonas aeruginosa arylsulfatase: a purified enzyme for the mild hydrolysis of steroid sulfates.

The hydrolysis of sulfate ester conjugates is frequently required prior to analysis for a range of analytical techniques including gas chromatography-mass spectrometry (GC-MS). Sulfate hydrolysis may be achieved with commercial crude arylsulfatase enzyme preparations such as that derived from Helix pomatia but these contain additional enzyme activities such as glucuronidase, oxidase, and reductase that make them unsuitable for many analytical applications. Strong acid can also be used to hydrolyze sulfate esters but this can lead to analyte degradation or increased matrix interference.

Compensatory stabilizing role of surface mutations during the directed evolution of dienelactone hydrolase for enhanced activity.

Directed evolution is a common tool employed to generate enzymes suitable for industrial use. High thermal stability is often advantageous or even a requirement for biocatalysts, as such the evolution of protein stability is of practical as well as academic interest. Even when evolving enzymes for new or improved catalytic functions, stability is an important factor since it can limit the accumulation rate and number of desired active site mutations.

Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search.

The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants.

Crystal structure of the mouse interleukin-3 β-receptor: insights into interleukin-3 binding and receptor activation.

Interleukin-3 (IL-3) is a cytokine secreted by mast cells and activated T-cells known to be an important regulator of differentiation, survival, proliferation and activation of a range of haemopoietic lineages. The effects of IL-3 on target cells are mediated by a transmembrane receptor system composed of a cytokine-specific α-subunit and a β-subunit, the principal signalling entity. In the mouse, two β-subunits have co-evolved: a common β-subunit (βc) shared between IL-3 and the related cytokines IL-5 and granulocyte/macrophage colony-stimulating factor (GM-CSF); and an IL-3-specific β-subunit (βIL-3).

Comparative investigation of the reaction mechanisms of the organophosphate-degrading phosphotriesterases from Agrobacterium radiobacter (OpdA) and Pseudomonas diminuta (OPH).

Metal ion-dependent, organophosphate-degrading enzymes have acquired increasing attention due to their ability to degrade and thus detoxify commonly used pesticides and nerve agents such as sarin. The best characterized of these enzymes are from Pseudomonas diminuta (OPH) and Agrobacterium radiobacter (OpdA). Despite high sequence homology (>90 % identity) and conserved metal ion coordination these enzymes display considerable variations in substrate specificity, metal ion affinity/preference and reaction mechanism.

Crystallization of dienelactone hydrolase in two space groups: structural changes caused by crystal packing.

Dienelactone hydrolase (DLH) is a monomeric protein with a simple α/β-hydrolase fold structure. It readily crystallizes in space group P2₁2₁2₁ from either a phosphate or ammonium sulfate precipitation buffer. Here, the structure of DLH at 1.

The Escherichia coli glucuronylsynthase promoted synthesis of steroid glucuronides: improved practicality and broader scope.

A library of steroid glucuronides was prepared using the glucuronylsynthase derived from Escherichia coliβ-glucuronidase, followed by purification using solid-phase extraction. A representative range of steroid substrates were screened for synthesis on the milligram scale under optimised conditions with conversions dependent on steroid substitution and stereochemistry. Epiandrosterone (3β-hydroxy-5α-androstan-17-one) provided the highest conversion of 90% (84% isolated yield).

Use of OpdA, an organophosphorus (OP) hydrolase, prevents lethality in an African green monkey model of acute OP poisoning.

Organophosphorus (OP) pesticides are a diverse class of acetylcholinesterase (AChE) inhibitors that are responsible for tremendous morbidity and mortality worldwide, killing approximately 300,000 people annually. Enzymatic hydrolysis of OPs is a potential therapy for acute poisoning. OpdA, an OP hydrolase isolated from Agrobacterium radiobacter, has been shown to decrease lethality in rodent models of OP poisoning.

Determination of the catalytic activity of binuclear metallohydrolases using isothermal titration calorimetry.

Binuclear metallohydrolases are a large and diverse family of enzymes that are involved in numerous metabolic functions. An increasing number of members find applications as drug targets or in processes such as bioremediation. It is thus essential to have an assay available that allows the rapid and reliable determination of relevant catalytic parameters (k cat, K m, and k cat/K m).

Immobilization of the enzyme GpdQ on magnetite nanoparticles for organophosphate pesticide bioremediation.

Annually thousands of people die or suffer from organophosphate (pesticide) poisoning. In order to remove these toxic compounds from the environment, the use of enzymes as bioremediators has been proposed. We report here a Ser127Ala mutant based on the enzyme glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes.

Spectroscopic and mechanistic studies of dinuclear metallohydrolases and their biomimetic complexes.

An enhanced understanding of the metal ion binding and active site structural features of phosphoesterases such as the glycerophosphodiesterase from Enterobacter aerogenes (GpdQ), and the organophosphate degrading agent from Agrobacterium radiobacter (OpdA) have important consequences for potential applications. Coupled with investigations of the metalloenzymes, programs of study to synthesise and characterise model complexes based on these metalloenzymes can add to our understanding of structure and function of the enzymes themselves. This review summarises some of our work and illustrates the significance and contributions of model studies to knowledge in the area.

Identification and characterization of an unusual metallo-β-lactamase from Serratia proteamaculans.

Metallo-β-lactamases (MBLs) are a family of metalloenzymes that are capable of hydrolyzing β-lactam antibiotics and are an important means by which bacterial pathogens use to inactivate antibiotics. A database search of the available amino acid sequences from Serratia proteamaculans indicates the presence of an unusual MBL. A full length amino acid sequence alignment indicates overall homology to B3-type MBLs, but also suggests considerable variations in the active site, notably among residues that are relevant to metal ion binding.

Structure and function of an insect α-carboxylesterase (αEsterase7) associated with insecticide resistance.

Insect carboxylesterases from the αEsterase gene cluster, such as αE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina (LcαE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the importance of OPs to agriculture and the spread of insect-borne diseases, the molecular basis for the ability of α-carboxylesterases to confer OP resistance to insects is poorly understood. In this work, we used laboratory evolution to increase the thermal stability of LcαE7, allowing its overexpression in Escherichia coli and structure determination.

In vitro directed evolution of enzymes expressed by E. coli in microtiter plates.

A method is described for using 96-well plates to prepare libraries of Escherichia coli cultures for screening a library of gene variants. This approach bypasses colony-picking to allow standard molecular biology laboratories to carry out directed evolution efficiently with a 96-well plate-reader and multichannel pipettes. Initial screens are applied to cultures that are rapidly prepared by diluting transformed cells so that an average of four cells starts each culture.

Screening libraries for improved solubility: using E. coli dihydrofolate reductase as a reporter.

Low protein solubility is a problem in many areas of protein science. Although chemical methods have been developed to solubilize proteins these are not always effective and add to the cost of producing the protein. One way of overcoming these difficulties is to evolve the protein to be more soluble.