Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610901
Title: Mechanism and stereoselectivity of N-acetylneuraminic acid lyase revealed by integrated experimental and computational studies
Author: Daniels , Adam David
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2012
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Abstract:
Enzymes are powerful biological catalysts and are of great interest in the chemical synthesis industry because of their ability to increase substantially reaction rates, control the stereochemical outcome of reactions and function in mild, environmentally friendly conditions. The effective creation of enzymes as industrial biocatalysts requires the engineering of tailored properties, using laboratory evolution or more sophisticated rational design methods. Rational enzyme design requires detailed structural and mechanistic information to be available. Computational methods, in particular QM/MM modelling, provide a way of accurately simulating enzyme reactions to elucidate catalytic mechanisms, contributing to the overall understanding of enzyme function. N-Acetylneuraminic acid lyase (NAL) is a Class I aldolase that catalyses the aldol addition between N-acetyl-D-mannosamine (ManNAc) and pyruvate to yield N-acetylneuraminic acid (Neu5Ac). 3-Fluoropyruvate is also a substrate for NAL variants, producing a variety of fluorinated sialic acid analogues (containing an additional stereocentre); these reactions were explored further using 19F-NMR spectroscopy. The Escherichia coli NAL (ecNAL) variant E192N shows a switch in specificity towards [2R,3S]-2,3-dihydroxy-4-oxo-N,N-dipropylbutanamide (DHOB) and displays poor stereoselectivity at (-4. QM/MM modelling of the wild-type and E192N ecNAL reactions (with the natural ,substrate pyruvate) has revealed valuable mechanistic information by identifying transition states, substrate binding conformations and important active site residues. The role of these key active site residues was subsequently probed in experimental studies. For the first time, strong evidence has been proposed to show that residue V137 is responsible for protonation of the aldehyde oxygen during the carbon-carbon bond formation. Furthermore, this study offers new evidence towards the explanation of the high stereoselectivity of wild-type ecNAL and the poor stereoselectivity of E192N ecNAL. This research provides insights into the structure-function relationship of enzymes and will allow for more informed decisions in the future rational design of ecNAL and other similar enzymes to engineer variants with tailored functionalities.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.610901  DOI: Not available
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