Goldman Peter J, Ryan Katherine S, Hamill Michael J, Howard-Jones Annaleise R, Walsh Christopher T, Elliott Sean J, Drennan Catherine L
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Chem Biol. 2012 Jul 27;19(7):855-65. doi: 10.1016/j.chembiol.2012.05.016.
The indolocarbazole biosynthetic enzymes StaC, InkE, RebC, and AtmC mediate the degree of oxidation of chromopyrrolic acid on route to the natural products staurosporine, K252a, rebeccamycin, and AT2433-A1, respectively. Here, we show that StaC and InkE, which mediate a net 4-electron oxidation, bind FAD with a micromolar K(d), whereas RebC and AtmC, which mediate a net 8-electron oxidation, bind FAD with a nanomolar K(d) while displaying the same FAD redox properties. We further create RebC-10x, a RebC protein with ten StaC-like amino acid substitutions outside of previously characterized FAD-binding motifs and the complementary StaC-10x. We find that these mutations mediate both FAD affinity and product specificity, with RebC-10x displaying higher StaC activity than StaC itself. X-ray structures of this StaC catalyst identify the substrate of StaC as 7-carboxy-K252c and suggest a unique mechanism for this FAD-dependent enzyme.
吲哚咔唑生物合成酶StaC、InkE、RebC和AtmC分别介导了通往天然产物星形孢菌素、K252a、瑞贝克霉素和AT2433 - A1的过程中铬吡咯酸的氧化程度。在此，我们表明，介导净4电子氧化的StaC和InkE以微摩尔级的解离常数（K(d)）结合黄素腺嘌呤二核苷酸（FAD），而介导净8电子氧化的RebC和AtmC以纳摩尔级的K(d)结合FAD，同时表现出相同的FAD氧化还原特性。我们进一步构建了RebC - 10x，这是一种在先前表征的FAD结合基序之外具有十个类似StaC氨基酸取代的RebC蛋白，以及互补的StaC - 10x。我们发现这些突变介导了FAD亲和力和产物特异性，其中RebC - 10x表现出比StaC本身更高的StaC活性。这种StaC催化剂的X射线结构确定StaC的底物为7 - 羧基 - K252c，并为这种依赖FAD的酶提出了一种独特的机制。
