Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603241
Title: Structure and function of an unusual heme dependent secondary amine oxidase
Author: Ortmayer, Mary
ISNI:       0000 0004 5355 6803
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2014
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Abstract:
The orphan enzyme “secondary amine monooxygenase (SAMO)” catalyses the N- dealkylation of dimethylamine (DMA) to methylamine and formaldehyde using NADPH and oxygen. It is described in the literature as a heme-dependent monooxygenase enzyme, similar in terms of the overall reaction catalysed to the well- characterised cytochrome P450s (CYPs). However, the SAMO heme is without the essential CYP thiolate ligand, featuring the more common histidine-ligation instead. During a study of bacterial operons containing H4F-binding modules, the genes encoding for SAMO were discovered. It was found that four genes αβγδ encode for the heterotetrameric SAMO. While sequence similarities indicate α encodes for an H4F- aminomethyltransferase module, and γ for an NADPH/FMN/2Fe2S reductase module, the function of β and δ genes was unclear. Spectral analysis of the Rhizobium etli CFN 42 δ-subunit reveals it to be the heme-binding domain (δRe), with the cofactor-free β- subunit responsible for enhanced DMA binding by the βδRe complex. DMA has the highest affinity for ferrous-oxy δRe, indicating close interaction between the dioxygen and DMA. Both δRe and βδRe have a positive redox potential supporting formation of a relatively stable dioxygen complex. Holoenzyme SAMO from Pseudomonas mendocina ymp (αβγδPm) demonstrates DMA dependent NADPH oxidation and formaldehyde production, but is inherently unstable. The crystal structure of SAMO heme domain from P. mendocina (δPm) reveals a heme-binding PAS domain. Capitalising on the 1.9 Å ternary complex structure with DMA and nitric oxide, a model of the SAMO catalytic mechanism was proposed. Conserved active site residues including Trp180 and Glu266 (involved in substrate binding), in addition to Arg224 were mutated, each rendering αβγδPm inactive. It is proposed that the positively charged amine substrate works in concert with conserved Arg224 residue to create a polarised active site, supporting heterolytic cleavage of the O-O bond. However, unlike other heme enzymes SAMO lacks a strong electronegative proximal ligand. In view of this, it is proposed SAMO catalyses an oxidase-type reaction, leading to a transient iminium species, in the absence of substrate oxygen insertion. To the best of current understanding, SAMO appears to be a novel type of PAS domain that performs an oxidase reaction with oxygen activation facilitated by the secondary amine substrate.
Supervisor: Munro, Andrew; Leys, David Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.603241  DOI: Not available
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