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首页> 外文期刊>Journal of Molecular Biology >Crystal structures of creatininase reveal the substrate binding site and provide an insight into the catalytic mechanism.
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Crystal structures of creatininase reveal the substrate binding site and provide an insight into the catalytic mechanism.

机译：肌酸酐酶的晶体结构揭示了底物结合位点，并提供了对催化机理的深入了解。

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摘要

Creatininase from Pseudomonas putida is a member of the urease-related amidohydrolase superfamily. The crystal structure of the Mn-activated enzyme has been solved by the single isomorphous replacement method at 1.8A resolution. The structures of the native creatininase and the Mn-activated creatininase-creatine complex have been determined by a difference Fourier method at 1.85 A and 1.6 A resolution, respectively. We found the disc-shaped hexamer to be roughly 100 A in diameter and 50 A in thickness and arranged as a trimer of dimers with 32 (D3) point group symmetry. The enzyme is a typical Zn2+ enzyme with a binuclear metal center (metal1 and metal2). Atomic absorption spectrometry and X-ray crystallography revealed that Zn2+ at metal1 (Zn1) was easily replaced with Mn2+ (Mn1). In the case of the Mn-activated enzyme, metal1 (Mn1) has a square-pyramidal geometry bound to three protein ligands of Glu34, Asp45, and His120 and two water molecules. Metal2 (Zn2) has a well-ordered tetrahedral geometry bound to the three protein ligands of His36, Asp45, and Glu183 and a water molecule. The crystal structure of the Mn-activated creatininase-creatine complex, which is the first structure as the enzyme-substrate/inhibitor complex of creatininase, reveals that significant conformation changes occur at the flap (between the alpha5 helix and the alpha6 helix) of the active site and the creatine is accommodated in a hydrophobic pocket consisting of Trp174, Trp154, Tyr121, Phe182, Tyr153, and Gly119. The high-resolution crystal structure of the creatininase-creatine complex enables us to identify two water molecules (Wat1 and Wat2) that are possibly essential for the catalytic mechanism of the enzyme. The structure and proposed catalytic mechanism of the creatininase are different from those of urease-related amidohydrolase superfamily enzymes. We propose a new two-step catalytic mechanism possibly common to creatininases in which the Wat1 acts as the attacking nucleophile in the water-adding step and the Wat2 actsas the catalytic acid in the ring-opening step.
机译：来自恶臭假单胞菌的肌酸酐酶是脲酶相关的酰胺水解酶超家族的成员。 Mn活化酶的晶体结构已通过单一同晶置换法以1.8A的分辨率进行了解析。天然肌酐酶和Mn激活的肌酐酶-肌酸复合物的结构已通过差示傅里叶法分别以1.85 A和1.6 A的分辨率确定。我们发现圆盘形六聚体的直径约为100 A，厚度约为50 A，并排列成具有32（D3）个点群对称性的二聚体三聚体。该酶是具有双核金属中心（metal1和metal2）的典型Zn2 +酶。原子吸收光谱法和X射线晶体学分析表明，金属1（Zn1）上的Zn2 +很容易被Mn2 +（Mn1）取代。对于Mn激活的酶，metal1（Mn1）具有与4个Glu34，Asp45和His120的蛋白质配体和2个水分子结合的方锥几何形状。 Metal2（Zn2）具有绑定到His36，Asp45和Glu183的三个蛋白质配体和一个水分子的有序四面体几何形状。 Mn激活的肌酐酶-肌酸复合物的晶体结构是肌酐酶的酶-底物/抑制剂复合物的第一个结构，显示出在其皮瓣（α5螺旋和α6螺旋之间）发生了显着的构象变化。活性位点和肌酸被容纳在由Trp174，Trp154，Tyr121，Phe182，Tyr153和Gly119组成的疏水口袋中。肌酸酐酶-肌酸复合物的高分辨率晶体结构使我们能够鉴定两个水分子（Wat1和Wat2），这对于酶的催化机制可能是必不可少的。肌酸酐酶的结构和拟议的催化机理不同于脲酶相关的酰胺水解酶超家族酶。我们提出了肌酸酐酶可能共有的新的两步催化机制，其中Wat1在加水步骤中充当攻击亲核体，Wat2在开环步骤中充当催化酸。

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