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Unraveling the roles of the reductant and free copper ions in LPMO kinetics
Biotechnology for Biofuels ( IF 6.3 ) Pub Date : 2021-01-21 , DOI: 10.1186/s13068-021-01879-0
Anton A. Stepnov , Zarah Forsberg , Morten Sørlie , Giang-Son Nguyen , Alexander Wentzel , Åsmund K. Røhr , Vincent G. H. Eijsink

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative depolymerization of industrially relevant crystalline polysaccharides, such as cellulose, in a reaction that depends on an electron donor and O2 or H2O2. While it is well known that LPMOs can utilize a wide variety of electron donors, the variation in reported efficiencies of various LPMO-reductant combinations remains largely unexplained. In this study, we describe a novel two-domain cellulose-active family AA10 LPMO from a marine actinomycete, which we have used to look more closely at the effects of the reductant and copper ions on the LPMO reaction. Our results show that ascorbate-driven LPMO reactions are extremely sensitive to very low amounts (micromolar concentrations) of free copper because reduction of free Cu(II) ions by ascorbic acid leads to formation of H2O2, which speeds up the LPMO reaction. In contrast, the use of gallic acid yields steady reactions that are almost insensitive to the presence of free copper ions. Various experiments, including dose–response studies with the enzyme, showed that under typically used reaction conditions, the rate of the reaction is limited by LPMO-independent formation of H2O2 resulting from oxidation of the reductant. The strong impact of low amounts of free copper on LPMO reactions with ascorbic acid and O2, i.e. the most commonly used conditions when assessing LPMO activity, likely explains reported variations in LPMO rates. The observed differences between ascorbic acid and gallic acid show a way of making LPMO reactions less copper-dependent and illustrate that reductant effects on LPMO action need to be interpreted with great caution. In clean reactions, with minimized generation of H2O2, the (O2-driven) LPMO reaction is exceedingly slow, compared to the much faster peroxygenase reaction that occurs when adding H2O2.

中文翻译:

揭示还原剂和游离铜离子在LPMO动力学中的作用

裂解多糖单加氧酶(LPMO)是一种单铜酶,可在取决于电子供体和O2或H2O2的反应中催化工业相关的结晶多糖(如纤维素)的氧化解聚。尽管众所周知,LPMO可以利用多种电子供体,但是各种LPMO-还原剂组合的报道效率的变化仍大体无法解释。在这项研究中,我们描述了一种来自海洋放线菌的新型两结构域纤维素活性家族AA10 LPMO,我们已使用它来更仔细地研究还原剂和铜离子对LPMO反应的影响。我们的结果表明,抗坏血酸盐驱动的LPMO反应对极少量(微摩尔浓度)的游离铜非常敏感,因为抗坏血酸还原游离Cu(II)离子会导致H2O2的形成,从而加速了LPMO反应。相反,没食子酸的使用产生稳定的反应,该反应对游离铜离子的存在几乎不敏感。各种实验,包括对该酶的剂量反应研究,表明在通常使用的反应条件下,反应速率受到还原剂氧化导致的LPMO非依赖性H2O2形成的限制。少量游离铜对LPMO与抗坏血酸和O2反应(即评估LPMO活性时最常用的条件)的强烈影响,可能解释了LPMO速率的变化。所观察到的抗坏血酸和没食子酸之间的差异显示出一种使LPMO反应对铜的依赖性降低的方法,并说明了对LPMO作用的还原剂作用需要格外谨慎。与添加H2O2时发生的更快的过氧合酶反应相比,在干净的反应中,H2O2的生成量最小,(O2驱动)的LPMO反应非常慢。
更新日期:2021-01-21
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