Cyanase is an inducible detoxifying enzyme in Escherichia coli that catalyzes decomposition of cyanate: OCN- + HCO3- + 2H+ --> 2CO2 + NH3 Cyanase is an octamer of identical subunits. The cyanase gene is part of a 3-gene operon that includes a permease for cyanate, cyanase, and a gene product of unknown function; the operon partially overlaps the lac operon, has been sequenced, and is controlled by a regulatory gene located only 108 base pairs upstream from the coding sequence. Important questions to be addressed in this study include 1) an explanation for the requirement of octameric structure of cyanase for catalytic activity and the utilization of bicarbonate as substrate (mechanism), 2) the mechanism, properties, and role of the permease, 3) the function and/or requirement of the third gene product of the cyanase operon, and 4) characterization of the repressor protein-DNA interaction. Site-specific mutagenesis and other biochemical genetic techniques which can be performed with relative ease, and the availability of cloned products of each of the 3 genes together with information about the structure of cyanase obtained by X-ray crystallography will greatly facilitate progress of these investigations. The results of this study will provide information about several fundamental biochemical processes including structure-function relationship of cyanase, role of oligomeric structure in a non-allosteric regulated enzyme, function and mechanism of an anion permease, and detailed properties of perhaps an unusual gene regulatory system. These studies may give insight into evolutionary aspects of this unusual operon. In addition, these studies may result n a direct health benefit. Cyanate administration as a drug or originating naturally from spontaneous breakdown of urea (present at high levels in uremia or chronic diarrhea) or as an environmental pollutant can have undesirable pharmacological effects. On the other hand, cyanate is a potential therapeutic agent, e.g. autotransfusion of cyanate-treated hemoglobin in sickle cell anemia, improving tolerance of blood cells to traumatic effects (surgery), and as an antitumor agent. Well characterized cyanase, particularly when immobilized, will be a useful reagent in these applications for analysis of cyanate or removal of cyanate where its presence is undesirable.
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