Louie G V, Brownlie P D, Lambert R, Cooper J B, Blundell T L, Wood S P, Malashkevich V N, Hädener A, Warren M J, Shoolingin-Jordan P M
Department of Crystallography, Birkbeck College, University of London, United Kingdom.
Proteins. 1996 May;25(1):48-78. doi: 10.1002/(SICI)1097-0134(199605)25:1<48::AID-PROT5>3.0.CO;2-G.
Porphobilinogen deaminase (PBGD) catalyses the polymerization of four molecules of porphobilinogen to form the 1-hydroxymethylbilane, preuroporphyrinogen, a key intermediate in the biosynthesis of tetrapyrroles. The three-dimensional structure of wild-type PBGD from Escherichia coli has been determined by multiple isomorphous replacement and refined to a crystallographic R-factor of 0.188 at 1.76 A resolution. the polypeptide chain of PBGD is folded into three alpha/beta domains. Domains 1 and 2 have a similar overall topology, based on a five-stranded, mixed beta-sheet. These two domains, which are linked by two hinge segments but otherwise make few direct interactions, form an extensive active site cleft at their interface. Domain 3, an open-faced, anti-parallel sheet of three strands, interacts approximately equally with the other two domains. The dipyrromethane cofactor is covalently attached to a cysteine side-chain borne on a flexible loop of domain 3. The cofactor serves as a primer for the assembly of the tetrapyrrole product and is held within the active site cleft by hydrogen-bonds and salt-bridges that are formed between its acetate and propionate side-groups and the polypeptide chain. The structure of a variant of PBGD, in which the methionines have been replaced with selenomethionines, has also been determined. The cofactor, in the native and functional form of the enzyme, adopts a conformation in which the second pyrrole ring (C2) occupies an internal position in the active site cleft. On oxidation, however, this C2 ring of the cofactor adopts a more external position that may correspond approximately to the site of substrate binding and polypyrrole chain elongation. The side-chain of Asp84 hydrogen-bonds the hydrogen atoms of both cofactor pyrrole nitrogens and also potentially the hydrogen atom of the pyrrole nitrogen of the porphobilinogen molecule bound to the proposed substrate binding site. This group has a key catalytic role, possibly in stabilizing the positive charges that develop on the pyrrole nitrogens during the ring-coupling reactions. Possible mechanisms for the processive elongation of the polypyrrole chain involve: accommodation of the elongating chain within the active site cleft, coupled with shifts in the relative positions of domains 1 and 2 to carry the terminal ring into the appropriate position at the catalytic site; or sequential translocation of the elongating polypyrrole chain, attached to the cofactor on domain 3, through the active site cleft by the progressive movement of domain 3 with respect to domains 1 and 2. Other mechanisms are considered although the amino acid sequence comparisons between PBGDs from all species suggest they share the same three-dimensional structure and mechanism of activity.
胆色素原脱氨酶（PBGD）催化四分子胆色素原聚合形成1-羟甲基胆素，即尿卟啉原，它是四吡咯生物合成中的关键中间体。通过多同晶置换法测定了来自大肠杆菌的野生型PBGD的三维结构，并在1.76 Å分辨率下精修至晶体学R因子为0.188。PBGD的多肽链折叠成三个α/β结构域。结构域1和2基于一个五链混合β折叠具有相似的整体拓扑结构。这两个结构域由两个铰链段相连，但除此之外几乎没有直接相互作用，它们在界面处形成一个宽阔的活性位点裂隙。结构域3是一个由三条链组成的开放型反平行片层，与其他两个结构域的相互作用大致相等。二吡咯甲烷辅因子共价连接到结构域3柔性环上的一个半胱氨酸侧链上。该辅因子作为四吡咯产物组装的引物，并通过其乙酸根和丙酸根侧基与多肽链之间形成的氢键和盐桥保持在活性位点裂隙内。还测定了PBGD的一个变体的结构，其中甲硫氨酸已被硒代甲硫氨酸取代。在该酶的天然功能形式中，辅因子采取一种构象，其中第二个吡咯环（C2）占据活性位点裂隙内的一个内部位置。然而，氧化时，该辅因子的C2环采取一个更外部的位置，这可能大致对应于底物结合和多吡咯链延伸的位点。Asp84的侧链与辅因子两个吡咯氮原子的氢原子形成氢键，也可能与结合到假定底物结合位点的胆色素原分子吡咯氮原子的氢原子形成氢键。该基团具有关键的催化作用，可能是在环偶联反应期间稳定吡咯氮原子上产生的正电荷。多吡咯链进行性延伸的可能机制包括：在活性位点裂隙内容纳延伸链，同时结构域1和2的相对位置发生移动，将末端环带到催化位点的适当位置；或者连接到结构域3上辅因子的延伸多吡咯链通过结构域3相对于结构域1和2的渐进移动依次穿过活性位点裂隙。尽管对来自所有物种的PBGD进行氨基酸序列比较表明它们具有相同的三维结构和活性机制，但也考虑了其他机制。
