Structure and function of an unusual heme dependent secondary amine oxidase

  • Mary Ortmayer

    Student thesis: Unknown


    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 alphaβγdelta encode for the heterotetrameric SAMO. While sequence similarities indicate alpha encodes for an H4F- aminomethyltransferase module, and γ for an NADPH/FMN/2Fe2S reductase module, the function of β and delta genes was unclear. Spectral analysis of the Rhizobium etli CFN 42 delta-subunit reveals it to be the heme-binding domain (deltaRe), with the cofactor-free β- subunit responsible for enhanced DMA binding by the βdeltaRe complex. DMA has the highest affinity for ferrous-oxy deltaRe, indicating close interaction between the dioxygen and DMA. Both deltaRe and βdeltaRe have a positive redox potential supporting formation of a relatively stable dioxygen complex. Holoenzyme SAMO from Pseudomonas mendocina ymp (alphaβγdeltaPm) demonstrates DMA dependent NADPH oxidation and formaldehyde production, but is inherently unstable. The crystal structure of SAMO heme domain from P. mendocina (deltaPm) 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 alphaβγdeltaPm 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.
    Date of Award1 Aug 2014
    Original languageEnglish
    Awarding Institution
    • The University of Manchester
    SupervisorDavid Leys (Supervisor) & Andrew Munro (Supervisor)

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