6YRA

Crystal structure of ATP-dependent caprolactamase from Pseudomonas jessenii

6YRA の概要

• エントリーDOI: 10.2210/pdb6yra/pdb
• 分子名称: Hydantoinase, 5-oxoprolinase, ZINC ION (3 entities in total)
• 機能のキーワード: caprolactam hydrolase, nylon 6 monomer, 6-aminocaproic acid, 5-oxoproline, phosphocaprolactam, carboxyphosphate, hydrolase
• 由来する生物種: Pseudomonas jessenii; 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 276571.70

構造登録者

Rozeboom, H.J.,Janssen, D.B. (登録日: 2020-04-20, 公開日: 2021-04-21, 最終更新日: 2024-11-06)

主引用文献

Marjanovic, A.,Rozeboom, H.J.,de Vries, M.S.,Mayer, C.,Otzen, M.,Wijma, H.J.,Janssen, D.B.
Catalytic and structural properties of ATP-dependent caprolactamase from Pseudomonas jessenii.
Proteins, 89:1079-1098, 2021

PubMed 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 (ΔG '= 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αβ) 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.

PubMed: 33826169
DOI: 10.1002/prot.26082

実験手法

X-RAY DIFFRACTION (4 Å)