Yeom H, Sligar S G, Li H, Poulos T L, Fulco A J
Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana 61801, USA.
Biochemistry. 1995 Nov 14;34(45):14733-40. doi: 10.1021/bi00045a014.
Cytochrome P450BM-3, a catalytically self-sufficient monooxygenase from Bacillus megaterium, catalyzes the omega-n (n = 1-3) hydroxylation of fatty acids in the presence of O2 and NADPH. Like most other P450s, cytochrome P450BM-3 contains a threonine residue (Thr268) in the distal I helix thought to be important for O2 binding and activation. Thr268 has been converted to alanine and the enzymatic properties and heme domain crystal structure determined. Using sodium laurate as the substrate, the mutant exhibited slower rates of O2 and NADPH consumption. In addition, electron transfer is uncoupled from substrate hydroxylation as evidenced by the greater production of water and peroxide in the mutant compared to the wild-type enzyme. The crystal structure of the mutant reveals that the only changes in structure are confined to the site of mutation. These data indicate an important role for Thr268 in O2 binding and activation in the metabolism of sodium laurate by cytochrome P450BM-3.
细胞色素P450BM-3是一种来自巨大芽孢杆菌的具有催化自足性的单加氧酶，在氧气和烟酰胺腺嘌呤二核苷酸磷酸（NADPH）存在的情况下，催化脂肪酸的ω-n（n = 1-3）羟基化反应。与大多数其他细胞色素P450一样，细胞色素P450BM-3在I螺旋远端含有一个苏氨酸残基（Thr268），该残基被认为对氧气的结合和活化很重要。已将Thr268转换为丙氨酸，并测定了其酶学性质和血红素结构域晶体结构。以月桂酸钠为底物时，该突变体的氧气和NADPH消耗速率较慢。此外，与野生型酶相比，突变体中产生的水和过氧化物更多，这表明电子传递与底物羟基化解偶联。突变体的晶体结构表明，结构上的唯一变化局限于突变位点。这些数据表明，Thr268在细胞色素P450BM-3代谢月桂酸钠的过程中，对氧气的结合和活化起着重要作用。
