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Title: Genetics and molecular mechanism of trimethylamine N-oxide demethylase of Methylocella silvestris BL2
Author: Zhu, Yijun
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2016
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Abstract:
Methylocella silvestris, an alphaproteobacterium isolated from a forest soil, can grow on trimethylamine N-oxide (TMAO) as a sole nitrogen source, however, the molecular and biochemical mechanisms underpinning its growth remain unknown. Marker-exchange mutagenesis enabled the identification of several genes involved in TMAO metabolism, including Msil_3606, a permease of the amino acids-polyamine (APC) superfamily, and Msil_3603, consisting a N-terminal domain of unknown function (DUF1989) and a C-terminal tetrahydrofolate-binding domain. Null mutants of Msil_3603 and Msil_3606 can no longer grow on TMAO. Purified Msil_3603 from recombinant Escherichia coli can convert TMAO to dimethylamine and formaldehyde (1 TMAO ! 1 dimethylamine + 1 formaldehyde), confirming that it encodes a bona fide TMAO demethylase (Tdm). Site-directed mutagenesis, homology modelling and metal analyses by inorganic mass spectrometry have been applied to gain insight into metal stoichiometry and underlying catalytic mechanism of Tdm of M. silvestris. Herein, it is demonstrated that active Tdm has 1 molar equivalent of Zn2+ and 1 molar equivalent of non-haem Fe2+. Further investigation of Zn2+ and Fe2+-binding sites through homology modelling and site-directed mutagenesis revealed that Zn2+ is coordinated by a 3- sulfur-1-O motif. An aspartate residue (D198) likely bridges Fe2+ and Zn2+ centres. H276, and maybe H256, contribute to Fe2+ binding. Site-directed mutagenesis of Tdm also led to the identification of three hydrophobic aromatic residues likely involved in substrate coordination (F259, Y305, W321), potentially through a cation-π interaction. Furthermore, a cross-over experiment using a substrate intermediate analogue gave direct evidence that a trimethylamine-alike intermediate was produced during the Tdm catalytic cycle, suggesting TMAO has a dual role of being both a substrate and an oxygen donor for formaldehyde formation. In this thesis, I attempted to resolve 3D-structure of Tdm to investigate structure-function relationship. Various Tdm homologues and mutants have been screened for crystallisation. Tdm from Ruegeria pomeroyi DSS-3 forms small 2D plates, hence warrants further refinements. Although Tdm of R.pomeroyi has comparable activity to that of M. silvestris, it is different from the one of M.silvestris in that a trimer and more susceptible to EDTA chelator. Together, this study has contributed to the understanding of the genetic and biochemical mechanisms for TMAO degradation in M. silvestris, and provides novel insight into the role of Zn2+ and Fe2+ in the catalysis of TMAO demethylation by this unique oxygen-independent enzyme.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.720490  DOI: Not available
Keywords: QR Microbiology
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