CLONING, EXPRESSION, PURIFICATION AND KINETIC STUDIES OF ESCHERICHIA COLI GMP REDUCTASE

Leonardo Martinelli (CPBMF INCT-TB PUCRS); Rodrigo Gay Ducati (CPBMF INCT-TB PUCRS); Luiz Augusto Basso (CPBMF INCT-TB PUCRS); Diógenes Santos (CPBMF INCT-TB PUCRS)

Guanosine monophosphate (GMP) reductase (NADPH: GMP oxireductase; EC 1.6.6.8) catalyzes the irreversible reductive deamination of GMP to inosine monophosphate (IMP), and is encoded by the guaC structural gene, playing an important role in the interconversion of purine nucleotides, particularly adenine and guanine. Thereby, this enzyme represents the only known metabolic step by which guanine nucleotides can be converted to the pivotal precursor of both adenine and guanine nucleotides. GMP reductase is not normally required for the efficient growth of Escherichia coli, since the lack of its activity leaves the de novo synthesis of both GMP and adenosine monophosphate (AMP) unimpaired; however, when the de novo synthesis of IMP is blocked, GMP reductase activity becomes necessary to supply AMP when guanine and its derivatives are the purine sources. The objectives of this work were to amplify and clone the guaC gene, and overexpress, purify and characterize the recombinant GMP reductase enzyme. The guaC structural gene (1038 bp) was PCR-amplified from E. coli K12 genomic DNA. The PCR product was cloned into pCR-Blunt vector and subcloned into pET-23a(+) expression vector, with NdeI and BamHI restriction sites. The resulting pET-23a(+)::guaC plasmid was sequenced to ensure gene integrity. The recombinant protein was expressed in the soluble form when the plasmid was transformed into E. coli BL21(DE3) host cells grown in LB medium. Three chromatographic steps were used to obtain GMP reductase in its homogenous form, and protein concentration was determined by the Bradford’s method. GMP reductase activity was determined spectrophotometrically by measuring the conversion of NADPH into NADP⁺ at 340 nm in order to determine the apparent kinetic constants (K_M, V_max, k_cat) for the catalyzed reaction. Future studies, such as initial velocity, product inhibition, and equilibrium binding will be important for the determination of the kinetic mechanism.