Co-expression of α and β subunits of the 3-methylcrotonyl-coenzyme A carboxylase from Pseudomonas aeruginosa.
Pseudomonas aeruginosa is a versatile bacterium that can grow using citronellol or leucine as sole carbon source. For both compounds the degradation pathways converge at the key enzyme 3-methylcrotonyl coenzyme-A carboxylase (MCCase). This enzyme is a complex formed by two subunits (α and β), encoded by the liuD and liuB genes, respectively; both are essential for enzyme function. Previously, both subunits had been separately expressed and then the complex re-constituted, however this methodology is laborious and produces low yield of active enzyme. In this work, the MCCase subunits were co-expressed in the same plasmid and purified in one step by affinity chromatography using the LiuD-His tag protein, interacting with the LiuB-S tag recombinant protein. The purified enzyme lost most of the activity within few hours of storage. The co-expressed subunits formed an (αβ)(4) complex that suffered a modification of its oligomerization state after storage, which probably contributed to the loss on activity observed. The recombinant MCCase enzyme presented optimum pH and temperature values of 9.0 and 30º C, respectively. Functionally, MCCase showed Michaelian kinetics behavior with a K(m) for its substrate and V(max) of 168 μM and 430 nmoles mg(-1)min(-1), respectively. The results suggest that the co-expression and co-purification of the subunits is a suitable procedure to obtain the active complex of the MCCase from Pseudomonas aeruginosa in a single step.
Instituto de Investigaciones Químico Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Edificio B-3, Ciudad Universitaria, 58030 Morelia, Michoacán, Mexico.
César
C
Díaz-Pérez
José Salud
JS
Rodríguez-Zavala
Alma Laura
AL
Díaz-Pérez
Jesús
J
Campos-García
D001709:Biotechnology / Q000379:methods; D019735:Carbon-Carbon Ligases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010957:Plasmids; D021122:Protein Subunits / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D021122:Protein Subunits; D011994:Recombinant Proteins; D019735:Carbon-Carbon Ligases; C021573:methylcrotonoyl-CoA carboxylase