Collyer CA

References (4)

Title : Compensatory stabilizing role of surface mutations during the directed evolution of dienelactone hydrolase for enhanced activity - Porter_2015_Protein.J_34_82
Author(s) : Porter JL , Collyer CA , Ollis DL
Ref : Protein J , 34 :82 , 2015
Abstract : 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. Dienelactone hydrolase, a small monomeric protein, has been previously evolved via a three-stage process to possess enhanced activity and specificity toward non-physiological substrates. In addition to seven active site mutations there were three surface mutations that were thought to increase the stability of the enzyme and compensate for the destabilizing active site mutations. Here, the individual influence of the three surface mutations--Q110L, Y137C and N154D--on the thermal and chemical stability of DLH has been assessed. While the Q110L and N154D mutations improved the thermal stability, the influence of the Y137C mutation was more complex. Individually it was destabilizing both thermally and chemically, but when in the presence of the Q110L and N154D mutations its effect was neutralized in relation to thermal but not chemical stability. In the context of a directed evolution experiment, these compensatory surface mutations play important roles. However, our results show that detrimental mutations can arise, thus the simultaneous monitoring of stability changes while evolving enzymes for enhanced catalytic properties can be beneficial.
ESTHER : Porter_2015_Protein.J_34_82
PubMedSearch : Porter_2015_Protein.J_34_82
PubMedID: 25600287

Title : Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search - Porter_2015_ACS.Chem.Biol_10_611
Author(s) : Porter JL , Boon PL , Murray TP , Huber T , Collyer CA , Ollis DL
Ref : ACS Chemical Biology , 10 :611 , 2015
Abstract : 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 alpha/beta 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. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.
ESTHER : Porter_2015_ACS.Chem.Biol_10_611
PubMedSearch : Porter_2015_ACS.Chem.Biol_10_611
PubMedID: 25419863
Gene_locus related to this paper: psepu-clcd1

Title : Crystallization of dienelactone hydrolase in two space groups: structural changes caused by crystal packing - Porter_2014_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_70_884
Author(s) : Porter JL , Carr PD , Collyer CA , Ollis DL
Ref : Acta Crystallographica F Struct Biol Commun , 70 :884 , 2014
Abstract : Dienelactone hydrolase (DLH) is a monomeric protein with a simple [alpha]/[beta]-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 1.85 A resolution crystallized in space group C2 with two molecules in the asymmetric unit is reported. When crystallized in space group P212121 DLH has either phosphates or sulfates bound to the protein in crucial locations, one of which is located in the active site, preventing substrate/inhibitor binding. Another is located on the surface of the enzyme coordinated by side chains from two different molecules. Crystallization in space group C2 from a sodium citrate buffer results in new crystallographic protein-protein interfaces. The protein backbone is highly similar, but new crystal contacts cause changes in side-chain orientations and in loop positioning. In regions not involved in crystal contacts, there is little change in backbone or side-chain configuration. The flexibility of surface loops and the adaptability of side chains are important factors enabling DLH to adapt and form different crystal lattices.
ESTHER : Porter_2014_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_70_884
PubMedSearch : Porter_2014_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_70_884
PubMedID: 25005082
Gene_locus related to this paper: psepu-clcd1

Title : Crystallization and preliminary crystallographic analysis of carboxypeptidase G2 from Pseudomonas sp. strain RS-16 - Lloyd_1991_J.Mol.Biol_220_17
Author(s) : Lloyd LF , Collyer CA , Sherwood RF
Ref : Journal of Molecular Biology , 220 :17 , 1991
Abstract : Carboxypeptidase G2, a zinc metalloenzyme isolated from Pseudomonas sp. strain RS-16, which catalyses the hydrolytic cleavage of reduced and non-reduced folates to pteroates and L-glutamate, has been crystallized from polyethylene glycol (average Mr 4000) by vapour diffusion. The crystal symmetry is monoclinic C2, with unit cell dimensions a = 206 A, b = 82 A, c = 116 A and beta = 118 degrees. The molecular mass and volume of the unit cell suggest that there are two dimers of the enzyme in the asymmetric unit. The crystals diffract to at least 3.0 A and are suitable for X-ray structure analysis.
ESTHER : Lloyd_1991_J.Mol.Biol_220_17
PubMedSearch : Lloyd_1991_J.Mol.Biol_220_17
PubMedID: 2067015