Research is proposed to develop insights into the mechanistic and evolutionary details of how biological systems use iron and manganese as biocatalytic centers in an overlapping set of enzymes. The focus of the research will be on a unique manganese-containing dioxygenase that is produced by Arthrobacter Mn-1. All other examples of 3,4- dihydroxyphenylacetate 2,3-dioxygenase that have been investigated with respect to metal content are known to contain iron. It is proposed to interface methodologies of molecular biology and biophysical chemistry to probe this iron and manganese dichotomy in oxygenase catalysis. A rapid enzyme purification method has been developed and some mechanistic experiments have been performed. The gene encoding the dioxygenase will be cloned to allow primary structure elucidation and site-specific mutagenesis to buttress biophysical approaches to probe the nature of the manganese ligands provided by the protein. Twenty-five additional bacteria which contain 3,4-dihydroxyphenylacetate 2,3- dioxygenase have been obtained which will allow comparisons in sequence and biophysical properties to derive further molecular insights. Native enzyme(s) will be examined by X-band and Q-band EPR and by extended X-ray absorption fine structure (EXAFS) methods. Additionally, 17O-substituted water, substrates, and inhibitors will be used with EPR and NMR to investigate events during the catalytic cycle of the manganese-containing dioxygenase. These techniques have previously proven effective in elucidating enzyme substrate interactions at the iron centers of the more well-studied non-heme iron dioxygenases. Efforts will be made to generate mutant enzymes to help unravel catalytic events and metal coordination by this unusual manganese-containing dioxygenase.
| Olson, P E; Qi, B; Que Jr, L et al. (1992) Immunological demonstration of a unique 3,4-dihydroxyphenylacetate 2,3-dioxygenase in soil Arthrobacter strains. Appl Environ Microbiol 58:2820-6 |
