Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan; Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-8628, Japan.
Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-8628, Japan.
J Inorg Biochem. 2021 Jun;219:111422. doi: 10.1016/j.jinorgbio.2021.111422. Epub 2021 Mar 15.
Dye-decolorizing peroxidase (DyP) is a heme-containing enzyme that catalyzes the degradation of anthraquinone dyes. A main feature of DyP is the acidic optimal pH for dye-decolorizing activity. In this study, we constructed several mutant DyP enzymes from Vibrio cholerae (VcDyP), with a view to identifying the decisive factor of the low pH preference of DyP. Initially, distal Asp144, a conserved residue, was replaced with His, which led to significant loss of dye-decolorizing activity. Introduction of His into a position slightly distant from heme resulted in restoration of activity but no shift in optimal pH, indicating that distal residues do not contribute to the pH dependence of catalytic activity. His178, an essential residue for dye decolorization, is located near heme and forms hydrogen bonds with Asp138 and Thr278. While Trp and Tyr mutants of His178 were inactive, the Phe mutant displayed ~35% activity of wild-type VcDyP, indicating that this position is a potential radical transfer route from heme to the active site on the protein surface. The Thr278Val mutant displayed similar enzymatic properties as WT VcDyP, whereas the Asp138Val mutant displayed significantly increased activity at pH 6.5. On the basis of these findings, we propose that neither distal amino acid residues, including Asp144, nor hydrogen bonds between His178 and Thr278 are responsible while the hydrogen bond between His178 and Asp138 plays a key role in the pH dependence of activity.
染料脱色过氧化物酶 (DyP) 是一种含有血红素的酶，可催化蒽醌染料的降解。DyP 的一个主要特征是用于染料脱色的最适酸性 pH。在这项研究中，我们构建了几种来自霍乱弧菌 (VcDyP) 的突变 DyP 酶，旨在确定 DyP 对低 pH 偏好的决定性因素。最初，保守残基远端 Asp144 被替换为 His，这导致染料脱色活性显著丧失。将 His 引入离血红素稍远的位置导致活性恢复，但最适 pH 没有变化，这表明远端残基不影响催化活性的 pH 依赖性。对染料脱色至关重要的残基 His178 位于血红素附近，与 Asp138 和 Thr278 形成氢键。虽然 His178 的 Trp 和 Tyr 突变体失活，但 Phe 突变体显示出野生型 VcDyP 的 ~35%活性，表明该位置是血红素到蛋白质表面活性位点的潜在自由基转移途径。Thr278Val 突变体显示出与 WT VcDyP 相似的酶学特性，而 Asp138Val 突变体在 pH 6.5 时显示出显著增加的活性。基于这些发现，我们提出远端氨基酸残基（包括 Asp144）和 His178 与 Thr278 之间的氢键都不是负责的因素，而 His178 与 Asp138 之间的氢键在活性的 pH 依赖性中起着关键作用。
