Pyruvate (Pyr) and α-ketoglutarate (αKg) accumulated when cells of Pseudomonas fluorescens NCIMB 11764 were cultivated on growth-limiting amounts of ammonia or cyanide and were shown to be responsible for the nonenzymatic removal of cyanide from culture fluids as previously reported (J.-L. Chen and D. A. Kunz, FEMS Microbiol. Lett. 156:61–67, 1997). The accumulation of keto acids in the medium paralleled the increase in cyanide-removing activity, with maximal activity (760 μmol of cyanide removed min−1 ml of culture fluid−1) being recovered after 72 h of cultivation, at which time the keto acid concentration was 23 mM. The reaction products that formed between the biologically formed keto acids and cyanide were unambiguously identified as the corresponding cyanohydrins by 13C nuclear magnetic resonance spectroscopy. Both the Pyr and α-Kg cyanohydrins were further metabolized by cell extracts and served also as nitrogenous growth substrates. Radiotracer experiments showed that CO2 (and NH3) were formed as enzymatic conversion products, with the keto acid being regenerated as a coproduct. Evidence that the enzyme responsible for cyanohydrin conversion is cyanide oxygenase, which was shown previously to be required for cyanide utilization, is based on results showing that (i) conversion occurred only when extracts were induced for the enzyme, (ii) conversion was oxygen and reduced-pyridine nucleotide dependent, and (iii) a mutant strain defective in the enzyme was unable to grow when it was provided with the cyanohydrins as a growth substrate. Pyr and αKg were further shown to protect cells from cyanide poisoning, and excretion of the two was directly linked to utilization of cyanide as a growth substrate. The results provide the basis for a new mechanism of cyanide detoxification and assimilation in which keto acids play an essential role.
In an attempt to see whether the C=O and the NH2 of CONH2 of asparagine5 glycinamide9 are both essential for biological activity, [5‐β‐cyanoalanine] oxytocin and [9‐α‐aminoacetonitrile] oxytocin have been synthesized. Each of these analogs contains a nitrile group in place of the carboxamide group of Asn5 GlyNH92 respectively; the nitrile group can simulate the carbonyl portion of the carboxamide, but lacks the hydrogen‐bond donating capacity of its NH2 portion. Substitution of a nitrile group produced opposite biological effects in the 5 and the 9 positions; the 5‐substituted analog showed very low activities (less than 3% of those of oxytocin) while the 9‐substituted analog showed extremely high activities (with an in vivo uterine activity of 906 U/mg almost twice that of oxytocin). The results clearly suggest that the mechanisms of interaction of the carboxamide groups with the receptor sites are different for residues 5 and 9.
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