Alternative routes of enzymic cyanide metabolism in Pseudomonas fluorescens NCIMB 11764.
Cell-free extracts from Pseudomonas fluorescens NCIMB 11764 catalysed the degradation of cyanide into products that included CO2, formic acid, formamide and ammonia. Cyanide-degrading activity (CDA) was localized to cytosolic cell fractions and was observed at substrate concentrations as high as 100 mM (2600 mg CN-I-1). At least two different CDAs could be distinguished by: (i) the determination of reaction product stoichiometries, (ii) requirements for NADH and oxygen, and (iii) kinetic analysis. The first activity produced CO2 and NH3 as reaction products, was dependent on oxygen and NADH for activity, and displayed an apparent Km for cyanide of 1.2 mM. The second activity generated formic acid (and NH3) plus formamide as reaction products, was oxygen independent, and had an apparent Km of 12 mM for cyanide. The first enzymic activity was identified as cyanide oxygenase as previously described [Harris, R. E. & Knowles, C. J. (1983) FEMS Microbiol Lett 20, 337-341] whereas the second activity is believed to consist of two enzymes, a cyanide nitrilase (dihydratase) and hydratase (EC 4.2.1.66). In addition to these enzymes, cyanide-grown cells were also induced for formate dehydrogenase (EC 1.2.1.2) thereby providing a means of recycling NADH utilized by cyanide oxygenase. A mutant strain having lost the ability to grow on cyanide as a nitrogen source was isolated and shown to be defective in cyanide oxygenase, but not the cyanide nitrilase/hydratase enzymes. This finding together with results showing that the substrate affinity of cyanide oxygenase was tenfold greater than for the nitrilase/hydratase enzymes, indicates that it is this enzyme that is most important in cyanide assimilation.(ABSTRACT TRUNCATED AT 250 WORDS)
Kunz
D A
DA
Department of Biological Sciences, University of North Texas, Denton 76203.
Wang
C S
CS
Chen
J L
JL
D000641:Ammonia / Q000032:analysis; D002245:Carbon Dioxide / Q000378:metabolism; D003486:Cyanides / Q000378:metabolism*; D005559:Formamides / Q000032:analysis; D005561:Formates / Q000032:analysis; D006836:Hydro-Lyases / Q000378:metabolism; D006867:Hydrolases / Q000378:metabolism; D008954:Models, Biological; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000378:metabolism
D003486:Cyanides; D005559:Formamides; D005561:Formates; D009243:NAD; C030544:formic acid; D002245:Carbon Dioxide; C031066:formamide; D000641:Ammonia; D010105:Oxygenases; D006867:Hydrolases; C083488:cyanide dihydratase; D006836:Hydro-Lyases; C076848:cyanide hydratase