Helin S, Kahn P C, Guha B L, Mallows D G, Goldman A
Turku Centre for Biotechnology, Finland.
J Mol Biol. 1995 Dec 15;254(5):918-41. doi: 10.1006/jmbi.1995.0666.
We report here the refined X-ray crystal structure of muconate lactonizing enzyme (MLE) from Pseudomonas putida PRS2000 at a resolution of 1.85 A with an R-factor of 16.8%. An enzyme from the beta-ketoadipate pathway, MLE catalyses the conversion of cis,cis-muconate to muconolactone. It is a homo-octamer, one monomer consisting of 373 amino acid residues. MLE has two large domains and a C-terminal subdomain: an alpha + beta domain, an alpha beta-barrel domain and a C-terminal meandering subdomain. The alpha beta-barrel domain is highly irregular. Its structure is (beta/alpha)7 beta, with the structural role of the last alpha-helix being replaced by both the C-terminal subdomain and part of the N-terminal domain. The fifth, seventh and eighth barrel strands are unusual because they have left-handed twist about their axes. The strand crossing angles also vary enormously, from +9 degrees to -69 degrees; the first and last strands, which close the barrel, cross at an angle of -69 degrees, making extensive strand-strand hydrogen bonding impossible. The first barrel strand is also unusual because it starts in the N-terminal domain and forms hydrogen bonds to the C-terminal subdomain beta-sheet as well as to its neighbouring strands in the barrel. It thus cements the whole protein together. As in other alpha beta-barrel proteins, the active site of MLE, present in each subunit is at the C-terminal ends of the barrel beta-strands. The active site cleft contains an essential manganese ion, is lined with charged and other polar residues, and contains many of the crystallographic water molecules. The manganese ion is octahedrally co-ordinated to three side-chain carboxylate groups and three water molecules, and is at the centre of a radiating web of ionic and hydrogen-bonding interactions. Additionally, two water molecules are buried in the centre of the barrel and two hydrophilic side-chains (Lys167 and Arg196) make both hydrophobic and hydrophilic packing interactions with much of the barrel interior. The barrel interior is thus also unusual because it is so hydrophilic; the dominating force appears to be the need to solvate the metal ion effectively. This might account for the irregularity of the barrel. The catalytic mechanism has been investigated by docking both substrate and product in the active site with the C-COO- of muconolactone superimposed on the corresponding atoms of cis,cis-muconate. In agreement with earlier kinetic and spectroscopic results, the manganese ion does not interact directly with substrate or product.(ABSTRACT TRUNCATED AT 400 WORDS)
我们在此报告恶臭假单胞菌PRS2000中粘康酸内酯化酶（MLE）的精细X射线晶体结构，分辨率为1.85 Å，R因子为16.8%。MLE是β-酮己二酸途径中的一种酶，催化顺，顺-粘康酸转化为粘康酸内酯。它是一种同八聚体，一个单体由373个氨基酸残基组成。MLE有两个大结构域和一个C端亚结构域：一个α + β结构域、一个αβ桶状结构域和一个C端蜿蜒亚结构域。αβ桶状结构域非常不规则。其结构为（β/α）7β，最后一个α螺旋的结构作用被C端亚结构域和N端结构域的一部分所取代。桶状结构的第五、第七和第八股链不寻常，因为它们围绕轴有左旋扭曲。链交叉角也有很大变化，从+9度到-69度；封闭桶状结构的第一股和最后一股链以-69度的角度交叉，使得广泛的链间氢键无法形成。第一股桶状链也不寻常，因为它起始于N端结构域，并与C端亚结构域β折叠以及桶状结构中相邻的链形成氢键。因此，它将整个蛋白质结合在一起。与其他αβ桶状蛋白质一样，MLE每个亚基中的活性位点位于桶状β链的C端。活性位点裂隙包含一个必需的锰离子，内衬带电和其他极性残基，并包含许多结晶水分子。锰离子以八面体方式与三个侧链羧酸盐基团和三个水分子配位，位于离子和氢键相互作用辐射网的中心。此外，两个水分子埋在桶状结构的中心，两个亲水性侧链（Lys167和Arg196）与桶状结构内部的大部分区域形成疏水和亲水堆积相互作用。因此，桶状结构内部也不寻常，因为它非常亲水；主要力量似乎是有效溶剂化金属离子的需要。这可能解释了桶状结构的不规则性。通过将底物和产物对接在活性位点上，并将粘康酸内酯的C-COO-与顺，顺-粘康酸的相应原子叠加，研究了催化机制。与早期的动力学和光谱结果一致，锰离子不直接与底物或产物相互作用。
