Chockalingam Karuppiah, Luba James, Nick Harry S, Silverman David N, Zhao Huimin
Department of Chemical Engineering and Biomolecular Engineering, Institute for Genomic Biology, Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
FEBS J. 2006 Nov;273(21):4853-61. doi: 10.1111/j.1742-4658.2006.05484.x. Epub 2006 Sep 22.
Human manganese superoxide dismutase is a mitochondrial metalloenzyme that is involved in protecting aerobic organisms against superoxide toxicity, and has been implicated in slowing tumor growth. Unfortunately, this enzyme exhibits strong product inhibition, which limits its potential biomedical applications. Previous efforts to alleviate human manganese superoxide dismutase product inhibition utilized rational protein design and site-directed mutagenesis. These efforts led to variants of human manganese superoxide dismutase at residue 143 with dramatically reduced product inhibition, but also reduced catalytic activity and efficiency. Here, we report the use of a directed evolution approach to engineer two variants of the Q143A human manganese superoxide dismutase mutant enzyme with improved catalytic activity and efficiency. Two separate activity-restoring mutations were found--C140S and N73S--that increase the catalytic efficiency of the parent Q143A human manganese superoxide dismutase enzyme by up to five-fold while maintaining low product inhibition. Interestingly, C140S is a context-dependent mutation, and the C140S-Q143A human manganese superoxide dismutase did not follow Michaelis-Menten kinetics. The re-engineered human manganese superoxide dismutase mutants should be useful for biomedical applications, and our kinetic and structural studies also provide new insights into the structure-function relationships of human manganese superoxide dismutase.
人锰超氧化物歧化酶是一种线粒体金属酶，参与保护需氧生物免受超氧化物毒性的影响，并与减缓肿瘤生长有关。不幸的是，这种酶表现出强烈的产物抑制作用，这限制了其潜在的生物医学应用。先前减轻人锰超氧化物歧化酶产物抑制作用的努力采用了合理的蛋白质设计和定点诱变。这些努力导致人锰超氧化物歧化酶在143位残基处的变体具有显著降低的产物抑制作用，但同时也降低了催化活性和效率。在此，我们报告使用定向进化方法设计出Q143A人锰超氧化物歧化酶突变酶的两个变体，其催化活性和效率得到了提高。发现了两个单独的活性恢复突变——C140S和N73S——它们将亲本Q143A人锰超氧化物歧化酶的催化效率提高了高达五倍，同时保持低产物抑制作用。有趣的是，C140S是一个依赖于上下文的突变，并且C140S-Q143A人锰超氧化物歧化酶不遵循米氏动力学。重新设计的人锰超氧化物歧化酶突变体应可用于生物医学应用，我们的动力学和结构研究也为人类锰超氧化物歧化酶的结构-功能关系提供了新的见解。
