Document Type

Theses, Ph.D


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Cinnamyl alcohol dehydrogenases (CAD; catalyse the conversion of p-hydroxy-cinnamaldehydes to their corresponding alcohols leading to the biosynthesis of lignin in plants. Outside of plants their role is less well defined. The cinnamyl alcohol dehydrogenase from H. pylori (HpCAD) has been cloned and produced in E. coli and characterised for substrate specificity. The enzyme is a monomer of 42.5 kDa found predominantly in the cytosol of the bacterium. It is specific for NADP(H) as cofactor and has a broad substrate specificity for alcohol and aldehyde substrates. Its substrate specificity is similar to the well-characterised plant enzymes. The best alcohol substrate was cinnamyl alcohol with a kcat/Km value of 126 S-1.m-1. The kcat/Km values for coniferyl alcohol and benzyl alcohol were more than an order of magnitude lower. Aliphatic alcohols were poorer substrates with kcat/Km values 10-fold or more lower than the aromatic alcohols. The kcat/Km values for aldehydes were higher than those for alcohols. Of the aromatic aldehydes, cinamylaldehyde was the best substrate followed by benzaldehyde. Acetaldehyde had a 10-fold lower kcat/Km value than cimmanylaldehyde. High substrate inhibition was observed and a mechanism of competitive inhibition proposed. The degree of high substrate inhibition was dependent on the substrate employed, for example aliphatic alcohols exhibited less pronounced inhibition than aromatics alcohols. No form of inhibition was evident even at 200 mM ethanol, while high substrate inhibition became apparent at 50 mM for both propanol and butanol. In contrast, all of the aromatic aldehyde substrates employed produced inhibition in the micro molar range; above 250 uM benzaldehyde, 150 uM cinnamaldehyde and 100 uM coniferyl aldehyde. The enzyme was found to be capable of catalysing the dismutation of benzaldehyde to benzyl alcohol and benzoic acid. This dismutation reaction has not previously been shown for this class of alcohol dehydrogenase and provides the bacterium with a means of reducing aldehyde concentration within the cell. An isogenic HpCAD negative mutant of H. pylori was also generated. The growth of the mutant both on plates and in liquid cultures was slower than the wild type. The growth of the mutant was further inhibited on exposure to mildly acidic conditions (pH 6.5 and 6.0).


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