Oxaloacetate Decarboxylase

Related Reading

Classifications

Oxaloacetate decarboxylase is divided into two types: cytoplasmic and plasma membrane types. The former does not have a coupled sodium ion transport function and was first identified in Microcococcus lysodeikticus.

Figure 1. Structure of oxaloacetate decarboxylase.

Role of oxaloacetate decarboxylase in the citric acid fermentation pathway

Citric acid is taken into the bacterium through coupled sodium ion export and is broken down to oxaloacetate and acetic acid by citrate lyase. Oxaloacetate is decarboxylated by oxaloacetate decarboxylase on the plasma membrane to produce pyruvate, while the decarboxylation reaction couples two sodium ions for export to the outside of the cell. Pyruvate is further broken down into acetyl coenzyme A and formic acid, followed by the reaction of acetyl coenzyme A to form acetyl phosphate. In this reaction pathway, it is particularly important for the biosynthetic reaction NADH synthesis.

Oxaloacetate decarboxylase subunits

Oxaloacetate decarboxylase consists of three different subunits α, β and λ in a 1:1:1 ratio. As shown in Figure 1, the peripheral α subunit (63.5 ku) contains two distinct structural domains, namely the N-terminal carboxyltransferase domain and the C-terminal biotin-binding domain. A proline- and alanine-rich peptide chain connects the biotin substitution between these two structural domains. At 35 amino acid residues from the C-terminus of the peptide chain, biotin is covalently attached to a lysine residue. The β-subunit of oxaloacetate decarboxylase (44.9 ku) is strongly hydrophobic and tightly bound to the plasma membrane.

Function of subunit

The oxaloacetate decarboxylase catalytic cycle begins with the binding of oxaloacetate to the α-subunit carboxyltransferase domain, followed by the transfer of the oxaloacetate carboxyl group to biotin, where the carboxylated biotin moves from the α-subunit carboxyltransferase site to the β-subunit decarboxylase site after a ductile proline alanine chain substitution. During the decarboxylation of carboxybiotin, an extra-membrane imported proton is depleted while two sodium ions are pumped into the periplasmic space. This series of biochemical reactions is closely related to the conformational changes of its protein. For example, replacement of the biotin component between the two catalytic structural domains may be responsible for opening and closing the sodium channels, and these conformational changes are spatially and temporally coordinated with the biochemical reactions and are critical for the overall decarboxylation reaction.

Purification

Oxaloacetate decarboxylase can be isolated and purified in a two-step extraction process, using a detergent to lyse the protein from the membrane, followed by affinity chromatography on an agarose column with the corresponding antibiotic protein. The three-subunit complex of Klebsiella pneumoniae oxaloacetate decarboxylase has been successfully synthesized in E. coli by induced expression, facilitating its study by mutagenesis, and the two-subunit complexes α_γ and β_γ have also been cloned, expressed and isolated in the experiment.

Membrane β-subunit topological isomerism analysis

Topological analysis of membrane proteins describes the number and orientation of transmembrane fragments and the C- and N-terminal distribution of a transmembrane protein. The β-subunit of oxaloacetate decarboxylase is a hydrophobic protein consisting of 433 amino acid residues, and hydrophobic analysis shows that it has 9 to 10 transmembrane structures. In the presumed transmembrane region, multiple sites were selected to interrupt the peptide chain of the target protein to form a fusion protein with alkaline phosphatase or β-galactosidase, and the entire protein was analyzed across the membrane by examining the location of the fusion site.