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Abstract：
Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence-related to other ATP-dependent enzymes involved in lactam hydrolysis, like 5-oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP-dependent acetone- and acetophenone carboxylases. The caprolactamase was produced in Escherichia coli, isolated by His-tag affinity chromatography, and subjected to functional and structural studies. Activity toward caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6-aminocaproic acid. Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG0 \'= 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αβ)2 tetramer and revealed an ATP-binding site in CapA and a Zn-coordinating site in CapB. Mutations in the ATP-binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A, and H124A) were inactive and less thermostable than the wild-type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP-dependent lactam hydrolysis.
摘要：
丙内酰胺酶是耶氏假单胞菌己内酰胺降解途径中的第一个酶。它由两个亚基（CapA和CapB）组成，并且与内酰胺水解中涉及的其他ATP依赖性酶相关，像5-氧代脯氨酸酶和乙内酰脲酶。ATP依赖性丙酮和苯乙酮羧化酶也存在低序列相似性。己内酰胺酶是在大肠杆菌中产生的，通过His-tag亲和层析分离，并进行功能和结构研究。对己内酰胺的活性需要ATP，并且取决于测定缓冲液中碳酸氢盐的存在。水解产物鉴定为6-氨基己酸。量子力学模型表明，己内酰胺的水解是非常不利的（ΔG0'=23kJ/mol），这解释了ATP的依赖性。晶体结构表明该酶以（αβ）2四聚体的形式存在，并揭示了CapA中的ATP结合位点和CapB中的Zn配位位点。CapA的ATP结合位点中的突变(D11A和D295A)显著减少产物形成。在CapB的金属结合位点（D41A，H99A,D101A,和H124A）无活性，热稳定性低于野生型酶。这些残基被证明对活性至关重要，根据实验结果，我们提出了ATP依赖性内酰胺水解的可能机制。
