The molecular analysis and biophysical characterisation of DMS dehydrogenase from Rhodovulum sulfidophilum

McDevitt, Christopher A. (2002). The molecular analysis and biophysical characterisation of DMS dehydrogenase from Rhodovulum sulfidophilum PhD Thesis, School of Molecular and Microbial Sciences, The University of Queensland.

       
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Author McDevitt, Christopher A.
Thesis Title The molecular analysis and biophysical characterisation of DMS dehydrogenase from Rhodovulum sulfidophilum
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
Publication date 2002
Thesis type PhD Thesis
Supervisor McEwan, Alastair
Total pages 357
Collection year 2002
Language eng
Subjects L
270000 Biological Sciences
780105 Biological sciences
Formatted abstract Dimethylsulfide (DMS) dehydrogenase is a heterotrimeric (aßy), b-type haem containing enzyme responsible for catalysing the oxidation of DMS to dimethylsulfoxide (DMSO) during the photoautotrophic growth of Rhodovulum sulfidophilum. Chemical analysis of purified DMS dehydrogenase showed that it contained approximately 0.65 mol Mo/mol enzyme, 6 mol P/mol enzyme, and 17 mol Fe/mol enzyme. Nucleotide analysis of DMS dehydrogenase identified bis(molybdopterin guanine dinucleotide)Mo, the form of the pterin molybdenum cofactor found in enzymes of the DMSO reductase family of molybdopterin-containing enzymes.

The operon encoding DMS dehydrogenase (ddhABDC) was cloned and sequenced. Sequence analysis of ddhA gene showed that it encoded a polypeptide with highest sequence similarity to the molybdopterin-containing subunits of selenate reductase (SerA), ethylbenzene dehydrogenase (EbdA) and respiratory nitrate reductase (NarG). Protein phylogenetic analysis indicated that these polypeptides formed a distinct clade within the DMSO reductase family of enzymes. Analysis of the primary structure of the DdhA polypeptide identified the four distinct structural domains as found in the crystal structures of enzymes of the DMSO reductase family. Multiple sequence alignment of the amino acid sequences of DdhA, SerA, NarG and EbdA showed that either a conserved threonine or serine residue could serve as a possible ligand to the Mo atom in this group of enzymes. ERR spectroscopy of the Mo(V) form of DMS dehydrogenase gave a typical rhombic spectrum with g-values (g1 = 1.9650, g2 = 1.9846, g3 = 2.0006) and indicated strong hyperfine coupling from a water molecule. Analysis of the rhombicity and anisotropy parameters of the Mo(V)-aqua spectrum of DMS dehydrogenase showed that it had highest similarity with the spectrum of the low pH nitrite Mo(V) species of nitrate reductase (NarGH). This was consistent with the close relationship between DMS dehydrogenase and respiratory nitrate reductase.

Further sequence analysis of the ddh gene cluster identified a potential iron-sulfur cluster-containing polypeptide DdhB. This polypeptide had high sequence similarity to NarH from respiratory nitrate reductase and contained the cysteine residues involved in the formation of three [4Fe-4S]2+,1+ and one [3Fe-4S]1+,0 clusters in NarH. ERR spectroscopy of DMS dehydrogenase identified the high potential [3Fe-4S]1+,0and [4Fe-4S]2+,1+clusters in the oxidised and reduced states respectively. Additional complexity upon further reduction indicated the presence of at least one or more additional iron-sulfur clusters. This is consistent with comparisons to the ß-subunit of respiratory nitrate reductase which has a similar organisation.

The ddh gene cluster also contained a gene determined to encode DdhC, the y-subunit of DMS dehydrogenase, which has been implicated in binding the b-type haem. Redox potentiometry and MOD spectroscopy confirmed the presence of a single low spin ferric haem with a midpoint potential of +315 mV and His-Met ligation. Mutational analysis identified the y-subunit as binding the haem. Conservation of the DdhC His and Met residues was observed in SerC and, thus, it is probable that the y-subunit selenate reductase also binds the b-type haem associated with that protein.

An additional gene was identified in ddh operon with no apparent corresponding protein present in the holoenzyme, a feature common in complex periplasmic multi-redox centre proteins. ddhD encodes a protein absent from purified DMS dehydrogenase and is suggested to be a cytoplasmic protein
Keyword Dehydrogenases.
Molecular biology.

 
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Created: Fri, 24 Aug 2007, 17:52:33 EST