Changes in the carboxyl terminus of the beta subunit of human propionyl-CoA carboxylase affect the oligomer assembly and catalysis: Expression and characterization of seven patient-derived mutant forms of PCC in Escherichia coli

Propionyl-CoA carboxylase (PCC) catalyzes the bioptin-dependent carboxylati on of propionyl-CoA to D-methylmalonyl-CoA in the mitochondrial matrix. Hum an PCC is a dodecamer composed of pairs of nonidentical alpha and beta subu nits encoded by PCCA and PCCB genes, respectively. Deficiency of PCC result s in propionic acidemia (PA), a metabolic disorder characterized by severe metabolic ketoacidosis, vomiting, lethargy, and hypotonia. To date, almost 60 mutations have been reported in both genes. Exon 15 of the beta subunit is one of the two sites where a number of mutations have been identified in PA patients. In the primary beta PCC sequence, these mutations lead to thr ee substitutions (R512C, L519P, and N536D), three truncations (R499X, R514X , and W531X), and one insertion (A513_R514insP). We expressed these mutant proteins in Escherichia coli in which the GroESL complex was overexpressed. The only mutation that does not impact the stability of mutant beta PCC in bacteria is W531X. The remaining mutations lead to either complete (L519P, N536D) or partial (R499X, R512C, A513_R514insP, and R514X) degradation of the mutant subunits. Size-exclusion chromatography revealed that R512C and W531X do not affect the assembly of alpha PCC and beta PCC to active oligom ers. Specific activities for these mutant proteins, however, were only 3.9 and 10% of the wild type, respectively. Taken together, the carboxyl-termin al portion of 40 amino acid residues of the beta subunit affects the stabil ity and the assembly of the alpha and beta subunits as well as the carboxyl ation of propionyl-CoA. (C) 2000 Academic Press.