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Applied and Environmental Microbiology, October 2008, p. 6280-6288, Vol. 74, No. 20
0099-2240/08/$08.00+0     doi:10.1128/AEM.00916-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of the Pseudomonas pseudoalcaligenes CECT5344 Cyanase, an Enzyme That Is Not Essential for Cyanide Assimilation{triangledown}

Víctor M. Luque-Almagro,1,{dagger} María-J. Huertas,1,{dagger} Lara P. Sáez,1 Manuel Martínez Luque-Romero,1 Conrado Moreno-Vivián,1 Francisco Castillo,1 M. Dolores Roldán,1 and Rafael Blasco2*

Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Edificio Severo Ochoa, 1a Planta, Campus de Rabanales, E-14071 Córdoba, Spain,1 Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura, Avenida de la Universidad SN, E-10071 Cáceres, Spain2

Received 23 April 2008/ Accepted 8 August 2008

Cyanase catalyzes the decomposition of cyanate into CO2 and ammonium, with carbamate as an unstable intermediate. The cyanase of Pseudomonas pseudoalcaligenes CECT5344 was negatively regulated by ammonium and positively regulated by cyanate, cyanide, and some cyanometallic complexes. Cyanase activity was not detected in cell extracts from cells grown with ammonium, even in the presence of cyanate. Nevertheless, a low level of cyanase activity was detected in nitrogen-starved cells. The cyn gene cluster of P. pseudoalcaligenes CECT5344 was cloned and analyzed. The cynA, cynB, and cynD genes encode an ABC-type transporter, the cynS gene codes for the cyanase, and the cynF gene encodes a novel {sigma}54-dependent transcriptional regulator which is not present in other bacterial cyn gene clusters. The CynS protein was expressed in Escherichia coli and purified by following a simple and rapid protocol. The P. pseudoalcaligenes cyanase showed an optimal pH of 8.5°C and a temperature of 65°C. An insertion mutation was generated in the cynS gene. The resulting mutant was unable to use cyanate as the sole nitrogen source but showed the same resistance to cyanate as the wild-type strain. These results, in conjunction with the induction pattern of the enzymatic activity, suggest that the enzyme has an assimilatory function. Although the induction of cyanase activity in cyanide-degrading cells suggests that some cyanate may be generated from cyanide, the cynS mutant was not affected in its ability to degrade cyanide, which unambiguously indicates that cyanate is not a central metabolite in cyanide assimilation.

* Corresponding author. Mailing address: Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura, Avenida de la Universidad SN, E-10071 Cáceres, Spain. Phone: 34 927257161. Fax: 34 92725710. E-mail: rblasco{at}unex.es

{triangledown} Published ahead of print on 15 August 2008.

{dagger} These authors contributed equally to this work.

Applied and Environmental Microbiology, October 2008, p. 6280-6288, Vol. 74, No. 20
0099-2240/08/$08.00+0     doi:10.1128/AEM.00916-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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