Acetyl Coenzyme A Synthetase (ADP Forming) from the Hyperthermophilic Archaeon Pyrococcus furiosus: Identification, Cloning, Separate Expression of the Encoding Genes, acdAI and acdBI, in Escherichia coli, and In Vitro Reconstitution of the Active Heterotetrameric Enzyme from Its Recombinant Subunits

Acetyl-coenzyme A (acetyl-CoA) synthetase (ADP forming) represents a novel enzyme in archaea of acetate formation and energy conservation (acetyl-CoA + ADP + P(i) → acetate + ATP + CoA). Two isoforms of the enzyme have been purified from the hyperthermophile Pyrococcus furiosus. Isoform I is a heterotetramer (α(2)β(2)) with an apparent molecular mass of 145 kDa, composed of two subunits, α and β, with apparent molecular masses of 47 and 25 kDa, respectively. By using N-terminal amino acid sequences of both subunits, the encoding genes, designated acdAI and acdBI, were identified in the genome of P. furiosus. The genes were separately overexpressed in Escherichia coli, and the recombinant subunits were reconstituted in vitro to the active heterotetrameric enzyme. The purified recombinant enzyme showed molecular and catalytical properties very similar to those shown by acetyl-CoA synthetase (ADP forming) purified from P. furiosus.

ADP形成型乙酰辅酶A（acetyl-CoA）合成酶是古菌中参与乙酸生成与能量守恒的新型酶类，其催化反应为：acetyl-CoA + ADP + P(i) → 乙酸 + ATP + 辅酶A。研究人员已从超嗜热菌激烈火球菌（Pyrococcus furiosus）中纯化得到该酶的两种同工型。其中同工型I为异四聚体(α₂β₂)，表观分子质量为145 kDa，由α、β两个亚基构成，二者的表观分子质量分别为47 kDa与25 kDa。通过分析两个亚基的N端氨基酸序列，我们在激烈火球菌的基因组中鉴定到其编码基因，分别命名为acdAI与acdBI。将这两个基因分别在大肠杆菌（Escherichia coli）中进行过表达，随后将所得重组亚基在体外重构为具有活性的异四聚体酶。纯化获得的重组酶所展现的分子特征与催化特性，与从激烈火球菌中纯化得到的天然ADP形成型乙酰辅酶A合成酶高度相似。