Field, M.; (2007) In vitro and in silico examination of the alternative respiratory NADH dehydrogenase family in Arabidopsis thaliana and other species. Doctoral thesis , University of London.

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Abstract

The respiratory electron transport chain (ETC) in plants, fungi and many bacteria incorporates additional enzymes not present in mammalian counterparts. The alternative NADH dehydrogenase (NDH2) performs the same enzymatic function as Complex I---the oxidation of NADU and reduction of membrane-bound quinone---but without the concomitant translocation of hydrogen ions across the ETC membrane, and thus does not contribute directly to the proton-motive potential. Three genes in Saccharomyces cerevisiae and as many as seven putative genes have been identified in Arabidopsis thaliana encoding NDH2 enzymes yet their structure, enzymatic kinetics and the benefits they confer on the organisms remain speculative. This thesis reports the investigation of these enzymes using a combination of computational and biochemical techniques. cDNA clones were produced from Arabidopsis leaf tissue and subcloned into an E. coli expression vector from which a high yield of protein was obtained, but this aggregated as inclusion bodies. Refolding assays using dialysis and injection diffusion techniques failed to obtain active protein. NDH2 gene sequences from all species were reviewed in detail both to determine expression patterns and to identify gene-specific motifs. The latter were used in a novel homology modelling process which has determined key structural elements, including residues specific for NADH or NADPH and the fact that these enzymes have been incorrectly annotated as "NADH disulphide oxidoreductases". The modelling analysis yielded other enzyme properties pertinent to biochemical expression, such as the absence of disulphide bridges in the tertiary structure.

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