Beinker Philipp, Lohkamp Bernhard, Peltonen Taina, Niemi Jarmo, Mäntsälä Pekka, Schneider Gunter
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm S-171 77, Sweden.
J Mol Biol. 2006 Jun 9;359(3):728-40. doi: 10.1016/j.jmb.2006.03.060. Epub 2006 Apr 19.
SnoaL2 and AclR are homologous enzymes in the biosynthesis of the aromatic polyketides nogalamycin in Streptomyces nogalater and cinerubin in Streptomyces galilaeus, respectively. Evidence obtained from gene transfer experiments suggested that SnoaL2 catalyzes the hydroxylation of the C-1 carbon atom of the polyketide chain. Here we show that AclR is also involved in the production of 1-hydroxylated anthracyclines in vivo. The three-dimensional structure of SnoaL2 has been determined by multi-wavelength anomalous diffraction to 2.5A resolution, and that of AclR to 1.8A resolution using molecular replacement. Both enzymes are dimers in solution and in the crystal. The fold of the enzyme subunits consists of an alpha+beta barrel. The dimer interface is formed by packing of the beta-sheets from the two subunits against each other. In the interior of the alpha+beta barrel a hydrophobic cavity is formed that most likely binds the substrate and harbors the active site. The subunit fold and the architecture of the active site in SnoaL2 and AclR are similar to that of the polyketide cyclases SnoaL and AknH; however, they show completely different quaternary structures. A comparison of the active site pockets of the putative hydroxylases AclR and SnoaL2 with those of bona fide polyketide cyclases reveals distinct differences in amino acids lining the cavity that might be responsible for the switch in chemistry. The moderate degree of sequence similarity and the preservation of the three-dimensional fold of the polypeptide chain suggest that these enzymes are evolutionary related. Members of this enzyme family appear to have evolved from a common protein scaffold by divergent evolution to catalyze reactions chemically as diverse as aldol condensation and hydroxylation.
SnoaL2和AclR分别是诺卡氏链霉菌中芳香族聚酮化合物诺加霉素生物合成以及加利利链霉菌中灰黄霉素生物合成过程中的同源酶。基因转移实验获得的证据表明，SnoaL2催化聚酮链C-1碳原子的羟基化反应。在此我们表明，AclR在体内也参与1-羟基化蒽环类药物的产生。已通过多波长反常衍射确定了SnoaL2的三维结构，分辨率为2.5埃，而AclR的三维结构则使用分子置换法确定，分辨率为1.8埃。这两种酶在溶液和晶体中均为二聚体。酶亚基的折叠结构由一个α+β桶状结构组成。二聚体界面是由两个亚基的β折叠相互堆积形成的。在α+β桶状结构的内部形成了一个疏水腔，该腔很可能结合底物并容纳活性位点。SnoaL2和AclR中，亚基折叠结构和活性位点的结构与聚酮环化酶SnoaL和AknH相似；然而，它们显示出完全不同的四级结构。将推定的羟化酶AclR和SnoaL2的活性位点口袋与真正的聚酮环化酶的活性位点口袋进行比较，发现腔内壁的氨基酸存在明显差异，这可能是导致化学反应转换的原因。适度的序列相似性以及多肽链三维折叠结构的保留表明这些酶在进化上是相关的。这个酶家族的成员似乎是通过趋异进化从一个共同的蛋白质支架进化而来，以催化诸如醛醇缩合和羟基化等化学性质不同的反应。
