Ohlendorf D H, Orville A M, Lipscomb J D
Department of Biochemistry, University of Minnesota Medical School Minneapolis 55455-0347.
J Mol Biol. 1994 Dec 16;244(5):586-608. doi: 10.1006/jmbi.1994.1754.
Protocatechuate 3,4-dioxygenase catalyzes the aromatic ring cleavage of 3,4-dihydroxybenzoate by incorporating both atoms of molecular oxygen to yield beta-carboxy-cis,cis-muconate. The structure of this metalloenzyme from Pseudomonas aeruginosa (now reclassified as P. putida) has been refined to an R-factor of 0.172 to 2.15 A resolution. The structure is a highly symmetric (alpha beta Fe3+)12 aggregate with a root-mean-square (r.m.s.) difference of 0.18 A among symmetry-related atoms. The tertiary structure of the two polypeptides (alpha and beta) are highly homologous (r.m.s. difference of 1.05 A over 127 C alpha atoms), suggesting that the ancestral enzyme was originally a homodimer with two active sites. Indeed, a non-functional, vestigial active site retains many of the properties of the functional active site but does not bind iron. The coordination geometry of the non-heme iron catalytic cofactor can best be described as trigonal bipyramidal with Tyr447 (147 beta) and His462 (162 beta) serving as axial ligands, and Tyr408 (108 beta), His460 (160 beta) and Wat837 serving as equitorial ligands. The active site environment has a number of basic residues that may promote binding of the acidic substrate. Within the putative active site cavity which is located between alpha and beta chains, five approximately coplanar solvent molecules suggest a position for the planar substrate Trp449 (149 beta), Ile491 (191 beta), defined by Gly14 (14 alpha) and Pro15 (15 alpha). In this position the guanidino group of Arg457 (157 beta) would be buried by the substrate, suggesting a functional role in catalysis.
原儿茶酸3,4 - 双加氧酶通过结合分子氧的两个原子来催化3,4 - 二羟基苯甲酸的芳香环裂解，生成β - 羧基 - 顺,顺 - 粘康酸。来自铜绿假单胞菌（现重新分类为恶臭假单胞菌）的这种金属酶的结构已被精修至R因子为0.172，分辨率为2.15 Å。该结构是一个高度对称的(αβFe3+)12聚集体，对称相关原子之间的均方根（r.m.s.）差异为0.18 Å。两条多肽链（α和β）的三级结构高度同源（在127个Cα原子上r.m.s.差异为1.05 Å），这表明原始酶最初是一个具有两个活性位点的同型二聚体。实际上，一个无功能的、残留的活性位点保留了许多功能活性位点的特性，但不结合铁。非血红素铁催化辅因子的配位几何结构最好描述为三角双锥，其中Tyr447（147β）和His462（162β）作为轴向配体，Tyr408（108β）、His460（160β）和Wat837作为赤道配体。活性位点环境有许多碱性残基，可能促进酸性底物的结合。在位于α链和β链之间的假定活性位点腔内，五个近似共面的溶剂分子表明平面底物Trp449（149β）、Ile491（191β）由Gly14（14α）和Pro15（15α）确定的位置。在这个位置，Arg457（157β）的胍基会被底物掩埋，这表明其在催化中起功能作用。
