Changes in the Carboxyl Terminus of the β 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

• Published in: Molecular genetics and metabolism Vol. 71; no. 4; pp. 623 - 632
• Main Authors: Chloupková, Maja, Ravn, Kirstine, Schwartz, Marianne, Kraus, Jan P.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-12-2000
• Subjects: Amino Acid Sequence; Base Sequence; Blotting, Western; Carboxy-Lyases - chemistry; Carboxy-Lyases - genetics; Carboxy-Lyases - metabolism; Catalysis; Chromatography, Gel; Enzyme Stability; Escherichia coli - genetics; Exons - genetics; Fibroblasts; Genotype; Humans; Metabolism, Inborn Errors - enzymology; Metabolism, Inborn Errors - genetics; Methylmalonyl-CoA Decarboxylase; mitochondria; Mitochondria - enzymology; Molecular Sequence Data; Mutation - genetics; mutations; Oligodeoxyribonucleotides; Phenotype; Propionates - blood; Propionates - metabolism; propionic acidemia; propionyl-CoA carboxylase; protein degradation; Protein Structure, Quaternary; Protein Subunits; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; propionic acidemia; mutations; propionyl-CoA carboxylase; mitochondria; protein degradation
• DOI: 10.1006/mgme.2000.3097
• EISSN: 1096-7206
• ISSN: 1096-7192

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 α and β 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 β subunit is one of the two sites where a number of mutations have been identified in PA patients. In the primary βPCC 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 βPCC 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 αPCC and βPCC 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 β subunit affects the stability and the assembly of the α and β subunits as well as the carboxylation of propionyl-CoA.