Cyanide is a notorious poison. Although highly toxic it represents a normal part of our environment for which mechanisms of biological generation (cyanogenesis) and degradation exist. While considerable information is available on enzymes responsible for cyanogenesis, much less is known about enzymes involved in cyanide turnover. The main objective of this project is to extend our knowledge about these enzymes by investigating the mechanisms used by bacteria for detoxifying and assimilating cyanide as a nutritional nitrogen source. Recent research has shown that in addition to the involvement of a novel enzyme (cyanide oxygenase CNO ), utilization of cyanide in the aerobic bacterium, Pseudomonas fluorescens, requires the excretion of (-keto acids (e.g., pyruvate). These findings have led to a two-step reaction model in which cyanide is first detoxified outside of the cell prior to being further assimilated within. The a-keto acids are believed to function as cyanide scavenging agents forming cyanohydrins as reaction products, and these compounds, rather than free cyanide (CN'/HCN), are thought to serve as enzymatic substrates for further assimilation. A fundamental objective of the project is to determine the mechanism by which the cyanohydrins are enzymatically metabolized. Research will be conducted both at UNT and at the University of Wisconsin, the latter while the PI is on a one-semester faculty development leave. The specific aims of the project are: (i) to clone and purify the CNO enzyme from P. fluorescens, (ii) to perform biochemical studies aimed at elucidating the reaction mechanism for CNO and enzymatic steps in cyanohydrin breakdown, and (iii) to isolate new anaerobic cyanide-utilizing bacteria for comparative studies with those ongoing in P. fluorescens. Work at UNT will focus on gene cloning and purification of the CNO enzyme using conventional molecular approaches. Research at UW will focus on learning new anaerobic cultivation techniques and will be conducted in collaborat ion with a resident anaerobic microbiologist (ARS, Dairy Forage Research Center). One objective of this collaborative effort will be to determine whether an anaerobic enzymatic counterpart to CNO can be shown to exist in bacteria that grow anaerobically on cyanide. Aside from basic new information on microbial enzymes and cyanide recycling in the biosphere, novel approaches for mitigating human and animal exposure from cyanide intoxication are expected. Related treatments for exposure from industrial contamination or deliberate release of cyanide as a genocidal agent are also anticipated.
