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Title: Structure, mechanism and engineering of pyridoxal phosphate-dependent racemases
Author: Frese, Amina
ISNI:       0000 0004 7431 9591
Awarding Body: University of York
Current Institution: University of York
Date of Award: 2018
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The objective of this work is the investigation and structural characterisation of pyridoxal phosphate (PLP)-dependent racemases. In particular α-amino-ε-caprolactam racemases (ACLRs), such as the enzyme from Achromobacter obae (AoACLR) are of interest for this work, as they have been found to racemise amino acid derivatives. This can be beneficial for the use of enzymes for the application for the dynamic kinetic resolution (DKR) of amino acid derivatives. In this work focus was put on the racemisation of the model substrate phenylalanine methylester due to the potential of a biocatalytic DKR of amino acid esters to the corresponding amides. In addition to AoACLR, two further enzymes were identified in a homology search. Racemases from Rhizobum freirei (RfACLR) and Ochrobactrum anthropi (OaACLR) were identified as targets for the evaluation of PLP-dependent racemases. These enzymes were heterologous expressed in Escherichia coli and purified. The purified ACLRs were subjected to crystallisation trials and especially RfACLR was found to crystallise well. Intensive structural analysis of RfACLR gave structures of different reaction intermediates of the racemisation reaction. A reaction mechanism proceeding via the formation of a geminal diamine and an achiral quinonoid was proposed based on these results. Furthermore, the catalytic residues D210 and K267 were identified by mutational analysis. The purified enzymes were subjected to activity assay to evaluate their substrate spectrum. Only OaACLR was found to be active towards phenylalanine methylester. With information obtained from the structure of OaACLR, structure-guided engineering of OaACLR resulted in 3.4-fold activity-improved variant. This variant, OaACLR-L293C, was characterised in detail and found to be an overall improved variant of OaACLR. OaACLR-L293C is therefore a promising biocatalyst for the application DKR of amino acid esters.
Supervisor: Grogan, Gideon Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available