Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, USA.
J Biol Chem. 2013 Jul 12;288(28):20668-82. doi: 10.1074/jbc.M113.479154. Epub 2013 Jun 3.
Steap4 is a cell surface metalloreductase linked to obesity-associated insulin resistance. Initial characterization of its cell surface metalloreductase activity has been reported, but thorough biochemical characterization of this activity is lacking. Here, we report detailed kinetic analysis of the Steap4 cell surface metalloreductase activities. Steap4 shows physiologically relevant Km values for both Fe(3+) and Cu(2+) and retains activity at acidic pH, suggesting it may also function within intracellular organelles to reduce these metals. Flavin-dependent NADPH oxidase activity that was much greater than the equivalent Steap3 construct was observed for the isolated N-terminal oxidoreductase domain. The crystal structure of the Steap4 oxidoreductase domain was determined, providing a structural explanation for these differing activities. Structure-function work also suggested Steap4 utilizes an interdomain flavin-binding site to shuttle electrons between the oxidoreductase and transmembrane domains, and it showed that the disordered N-terminal residues do not contribute to enzymatic activity.
Steap4 是一种与肥胖相关的胰岛素抵抗相关的细胞表面金属还原酶。已经报道了其细胞表面金属还原酶活性的初步特征，但这种活性的彻底生化特征尚不清楚。在这里，我们报告了 Steap4 细胞表面金属还原酶活性的详细动力学分析。Steap4 对 Fe(3+)和 Cu(2+)均表现出生理相关的 Km 值，并且在酸性 pH 下保持活性，这表明它也可能在细胞内细胞器中发挥作用，以还原这些金属。与相当的 Steap3 构建体相比，观察到分离的 N 端氧化还原酶结构域具有更高的黄素依赖性 NADPH 氧化酶活性。Steap4 氧化还原酶结构域的晶体结构已被确定，为这些不同的活性提供了结构解释。结构功能研究还表明，Steap4 利用一个结构域间黄素结合位点在氧化还原酶和跨膜结构域之间传递电子，并且表明无序的 N 端残基不贡献于酶活性。
