Thermodynamic and mutational studies of l-N-carbamoylase from Sinorhizobium meliloti CECT 4114 catalytic centre

Biochimie
Sergio Martínez-RodríguezFrancisco Javier Las Heras-Vázquez

Abstract

Purified site-directed mutants of Sinorhizobium meliloti CECT 4114 l-N-carbamoylase (SmLcar) in which Glu132, His230, Asn279 and Arg292 were replaced have been studied by kinetic methods and isothermal titration calorimetry (ITC). The importance of His230, Asn279 and Arg292 residues in the recognition of N-carbamoyl-l-alpha-amino acids has been proved. The role of Glu132 has been confirmed in substrate hydrolysis. ITC has confirmed two Ni atoms per monomer of wild type enzyme, and two equal and independent substrate binding sites (one per monomer). Homology modelling of SmLcar supports the importance of His87, His194, His386, Glu133 and Asp98 in metal binding. A comprehensive reaction mechanism is proposed on the basis of binding experiments measured by ITC, kinetic assays, and homology of the active centre with beta-alanine synthase from Saccharomyces kluyveri and other enzymes.

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Citations

Oct 16, 2009·Applied Microbiology and Biotechnology·Sergio Martínez-RodríguezFrancisco Javier Las Heras-Vázquez
Dec 5, 2008·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Sergio Martínez-RodríguezJose Antonio Gavira
Nov 18, 2008·Applied and Environmental Microbiology·Ana Isabel Martínez-GómezFrancisco Javier Las Heras-Vázquez
Aug 21, 2012·Journal of Bacteriology·Sergio Martínez-RodríguezJose Antonio Gavira
Jan 18, 2008·Journal of Molecular Recognition : JMR·Oksana Okhrimenko, Ilian Jelesarov
Mar 18, 2008·Biochemistry·Holger A LindnerTraian Sulea

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