Johjima T, Itoh N, Kabuto M, Tokimura F, Nakagawa T, Wariishi H, Tanaka H
Department of Forest Products, Kyushu University, Fukuoka 812-8581, Japan.
Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):1989-94. doi: 10.1073/pnas.96.5.1989.
Binding properties of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium against a synthetic lignin (dehydrogenated polymerizate, DHP) were studied with a resonant mirror biosensor. Among several ligninolytic enzymes, only LiP specifically binds to DHP. Kinetic analysis revealed that the binding was reversible, and that the dissociation equilibrium constant was 330 microM. The LiP-DHP interaction was controlled by the ionization group with a pKa of 5.3, strongly suggesting that a specific amino acid residue plays a role in lignin binding. A one-electron transfer from DHP to oxidized intermediates LiP compounds I and II (LiPI and LiPII) was characterized by using a stopped-flow technique, showing that binding interactions of DHP with LiPI and LiPII led to saturation kinetics. The dissociation equilibrium constants for LiPI-DHP and LiPII-DHP interactions were calculated to be 350 and 250 microM, and the first-order rate constants for electron transfer from DHP to LiPI and to LiPII were calculated to be 46 and 16 s-1, respectively. These kinetic and spectral studies strongly suggest that LiP is capable of oxidizing lignin directly at the protein surface by a long-range electron transfer process. A close look at the crystal structure suggested that LiP possesses His-239 as a possible lignin-binding site on the surface, which is linked to Asp-238. This Asp residue is hydrogen-bonded to the proximal His-176. This His-Asp...proximal-His motif would be a possible electron transfer route to oxidize polymeric lignin.
利用共振镜生物传感器研究了担子菌黄孢原毛平革菌中木质素过氧化物酶（LiP）与合成木质素（脱氢聚合物，DHP）的结合特性。在几种木质素分解酶中，只有LiP能特异性结合DHP。动力学分析表明这种结合是可逆的，解离平衡常数为330微摩尔。LiP-DHP相互作用受pKa为5.3的离子化基团控制，强烈表明特定氨基酸残基在木质素结合中起作用。利用停流技术表征了从DHP到氧化中间体LiP化合物I和II（LiPI和LiPII）的单电子转移，表明DHP与LiPI和LiPII的结合相互作用导致饱和动力学。LiPI-DHP和LiPII-DHP相互作用的解离平衡常数经计算分别为350和250微摩尔，从DHP到LiPI和到LiPII的电子转移一级速率常数经计算分别为46和16 s-1。这些动力学和光谱研究强烈表明LiP能够通过远程电子转移过程在蛋白质表面直接氧化木质素。仔细观察晶体结构表明LiP在表面具有His-239作为可能的木质素结合位点，它与Asp-238相连。这个Asp残基与近端His-176形成氢键。这种His-Asp...近端-His基序可能是氧化聚合木质素的电子转移途径。
