Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.771294
Title: Halogenases for biosynthesis and biocatalysis
Author: Chinnan Velmurugan, Karthikeyan
ISNI:       0000 0004 7657 3753
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2014
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Abstract:
Halogenation is an important chemical and biological process in the production of industrially important halogenated compounds. The presence of the halogen enhances the desirable properties of many of these compounds. However, production of halogenated compounds chemically often generates by-products that cause environmental pollution and the reactions often lack regio/stereo-selectivity. Biocatalysis have emerged to overcome these issues providing us with a novel technique to produce much greener regio-selective halogenated compounds. The first project involved the study of putative halogenase Ram20 from the ramoplanin biosynthetic gene cluster which is predicted to chlorinate the non-proteinogenic amino acid Hydroxyphenylglycine (Hpg) found at position 17 of ramoplanin. We tried to develop an in vitro system which would allow us to investigate Ram20's ability to chlorinate Hpg-17. To achieve this we expressed the peptidyl carrier protein (PCP) domain of the ramoplanin non-ribosomal synthetases module to which Hpg-17 is tethered, a phosphopantethenyl transferase Sfp required to load the HPG onto the PCP domain and Ram20. Synthetic compounds CoA-Hpg and CoA-SNAC were synthesised separately. Unfortunately, no evidence for chlorination of Hpg could be obtained. The second project involved the study of tryptophan halogenases, with the focus on the flavin dependent halogenases that regio-selectively chlorinate small substrates. The flavin dependent halogenases PrnA, KtzQ, SttH, KtzR and PyrH were produced in E. coli for the first time. The enzymes were assayed with tryptophan and a wider range of aromatic and phenolic substrates. KtzQ failed to show any promising activity, while KtzR could only chlorinate tryptophan. The other halogenases, PrnA, SttH and PyrH, were able to chlorinate a wider range of substrates including tryptophan, kynurenine, anthranilamide, 2-amino-4-methylbenzamide and anthranilic acid. Mutagenesis was performed on PrnA to widen the substrate scope, wherein a mutant F454K was identified that had higher halogenation activity with anthranilic acid compared to that of the wild type. The crystal structure of this mutant bound with FAD and Cl- was obtained. Work was also carried out to identify important residues that govern regio-selective halogenation of these enzymes. In particular, attempts were made to obtain a crystal structure of SttH, a tryptophan-6-halogenase to study the mechanism of regio-selective halogenation at the 6-position.
Supervisor: Micklefield, Jason Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.771294  DOI: Not available
Keywords: halogenation ; biocatalysis ; biosynthesis
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