Forest K T, Langford P R, Kroll J S, Getzoff E D
Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, Mail Drop MB-4, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
J Mol Biol. 2000 Feb 11;296(1):145-53. doi: 10.1006/jmbi.1999.3448.
Macrophages and neutrophils protect animals from microbial infection in part by issuing a burst of toxic superoxide radicals when challenged. To counteract this onslaught, many Gram-negative bacterial pathogens possess periplasmic Cu,Zn superoxide dismutases (SODs), which act on superoxide to yield molecular oxygen and hydrogen peroxide. We have solved the X-ray crystal structure of the Cu,Zn SOD from Actinobacillus pleuropneumoniae, a major porcine pathogen, by molecular replacement at 1.9 A resolution. The structure reveals that the dimeric bacterial enzymes form a structurally homologous class defined by a water-mediated dimer interface, and share with all Cu,Zn SODs the Greek-key beta-barrel subunit fold with copper and zinc ions located at the base of a deep loop-enclosed active-site channel. Our structure-based sequence alignment of the bacterial enzymes explains the monomeric nature of at least two of these, and suggests that there may be at least one additional structural class for the bacterial SODs. Two metal-mediated crystal contacts yielded our C222(1) crystals, and the geometry of these sites could be engineered into proteins recalcitrant to crystallization in their native form. This work highlights structural differences between eukaryotic and prokaryotic Cu,Zn SODs, as well as similarities and differences among prokaryotic SODs, and lays the groundwork for development of antimicrobial drugs that specifically target periplasmic Cu,Zn SODs of bacterial pathogens.
巨噬细胞和中性粒细胞在受到挑战时会释放出一阵有毒的超氧自由基，从而在一定程度上保护动物免受微生物感染。为了抵御这种攻击，许多革兰氏阴性细菌病原体都拥有周质铜锌超氧化物歧化酶（SOD），该酶作用于超氧化物，产生分子氧和过氧化氢。我们通过分子置换法以1.9埃的分辨率解析了胸膜肺炎放线杆菌（一种主要的猪病原体）的铜锌SOD的X射线晶体结构。该结构表明，二聚体细菌酶形成了一个由水介导的二聚体界面定义的结构同源类，并且与所有铜锌SOD共享希腊钥匙β桶亚基折叠，铜和锌离子位于深环封闭活性位点通道的底部。我们基于结构的细菌酶序列比对解释了其中至少两种酶的单体性质，并表明细菌SOD可能至少还有一种额外的结构类。两种金属介导的晶体接触产生了我们的C222(1)晶体，并且这些位点的几何结构可以被设计到以天然形式难以结晶的蛋白质中。这项工作突出了真核和原核铜锌SOD之间的结构差异，以及原核SOD之间的异同，并为开发专门针对细菌病原体周质铜锌SOD的抗菌药物奠定了基础。
