Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland.
Biochemistry. 2010 Feb 9;49(5):934-44. doi: 10.1021/bi901985b.
SnoaB is a cofactor-independent monooxygenase that catalyzes the conversion of 12-deoxynogalonic acid to nogalonic acid in the biosynthesis of the aromatic polyketide nogalamycin in Streptomyces nogalater. In vitro (18)O(2) experiments establish that the oxygen atom incorporated into the substrate is derived from molecular oxygen. The crystal structure of the enzyme was determined in two different space groups to 1.7 and 1.9 A resolution, respectively. The enzyme displays the ferredoxin fold, with the characteristic beta-strand exchange at the dimer interface. The crystal structures reveal a putative catalytic triad involving two asparagine residues, Asn18 and Asn63, and a water molecule, which may play important roles in the enzymatic reaction. Site-directed mutagenesis experiments, replacing the two asparagines individually by alanine, led to a 100-fold drop in enzymatic activity. Replacement of an invariant tryptophan residue in the active site of the enzyme by phenylalanine also resulted in an enzyme variant with about 1% residual activity. Taken together, our findings are most consistent with a carbanion mechanism where the deprotonated substrate reacts with molecular oxygen via one electron transfer and formation of a caged radical.
SnoaB 是一种辅因子非依赖性单加氧酶，它在 Streptomyces nogalater 中芳香聚酮类诺加霉素的生物合成中催化 12-去氧葡萄糖酸向诺加酸的转化。体外 (18)O(2) 实验确定掺入底物的氧原子来自于分子氧。该酶的晶体结构分别在两个不同的空间群中解析到 1.7 和 1.9Å分辨率。该酶呈现出铁氧还蛋白折叠结构，在二聚体界面处具有特征性的β-链交换。晶体结构揭示了一个可能的催化三联体，涉及两个天冬酰胺残基，Asn18 和 Asn63，以及一个水分子，它们可能在酶反应中发挥重要作用。定点突变实验，将两个天冬酰胺残基分别突变为丙氨酸，导致酶活性下降 100 倍。酶活性位点中一个不变色氨酸残基被苯丙氨酸取代，也导致酶变体的活性残留约 1%。总的来说，我们的发现最符合碳负离子机制，其中去质子化的底物通过单电子转移和形成笼状自由基与分子氧反应。
