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 biotin-dependent carboxylation of propionyl-CoA to d-methylmalonyl-CoA in the mitochondrial matrix. Human PCC is a dodecamer composed of pairs of nonidentical alpha and beta subunits encoded by PCCA and PCCB genes, respectively. Deficiency of PCC results 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 betaPCC sequence, these mutations lead to three substitutions (R512C, L519P, and N536D), three truncations (R499X, R514X, and W531X), and one insertion (A51_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 betaPCC 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 alphaPCC and betaPCC to active oligomers. Specific activities for these mutant proteins, however, were only 3.9 and 10% of the wild type, respectively. Taken together, the carboxyl-terminal portion of 40 amino acid residues of the beta subunit affects the stability and the assembly of the alpha and beta subunits as well as the carboxylation of propionyl-CoA.