Antson A A, Demidkina T V, Gollnick P, Dauter Z, von Tersch R L, Long J, Berezhnoy S N, Phillips R S, Harutyunyan E H, Wilson K S
Shubnikov Institute of Crystallography, Russian Academy of Sciences, Moscow.
Biochemistry. 1993 Apr 27;32(16):4195-206. doi: 10.1021/bi00067a006.
Tyrosine phenol-lyase (EC 4.1.99.2) from Citrobacter freundii has been cloned and the primary sequence deduced from the DNA sequence. From the BrCN digest of the NaBH4-reduced holoenzyme, five peptides were purified and sequenced. The amino acid sequences of the peptides agreed with the corresponding parts of the tyrosine phenol-lyase sequence obtained from the gene structure. K257 is the pyridoxal 5'-phosphate binding residue. Assisted by the sequence data, the crystal structure of apotyrosine phenol-lyase, a pyridoxal 5'-phosphate-dependent enzyme, has been refined to an R-factor of 16.2% at 2.3-A resolution using synchrotron radiation diffraction data. The tetrameric molecule has 222 symmetry, with one of the axes coincident with the crystallographic 2-fold symmetry axis of the crystal which belongs to the space group P2(1)2(1)2 with a = 76.0 A, b = 138.3 A, and c = 93.5 A. Each subunit comprises 14 alpha-helices and 16 beta-strands, which fold into a small and a large domain. The coenzyme-binding lysine residue is located at the interface between the large and small domains of one subunit and the large domain of a crystallographically related subunit. The fold of the large, pyridoxal 5'-phosphate binding domain and the location of the active site are similar to that found in aminotransferases. Most of the residues which participate in binding of pyridoxal 5'-phosphate in aminotransferases are conserved in the structure of tyrosine phenol-lyase. Two dimers of tyrosine phenol-lyase, each of which has a domain architecture similar to that found in aspartate aminotransferases, are bound together through a hydrophobic cluster in the center of the molecule and intertwined N-terminal arms.
来自弗氏柠檬酸杆菌的酪氨酸酚裂解酶（EC 4.1.99.2）已被克隆，并从DNA序列推导得到其一级序列。通过对硼氢化钠还原的全酶进行溴化氰消化，纯化并测序了五个肽段。这些肽段的氨基酸序列与从基因结构获得的酪氨酸酚裂解酶序列的相应部分一致。K257是磷酸吡哆醛结合残基。在序列数据的辅助下，使用同步辐射衍射数据，将一种磷酸吡哆醛依赖性酶——脱辅基酪氨酸酚裂解酶的晶体结构精修至2.3埃分辨率下R因子为16.2%。四聚体分子具有222对称性，其中一个轴与属于空间群P2(1)2(1)2（a = 76.0埃，b = 138.3埃，c = 93.5埃）的晶体的结晶学2重对称轴重合。每个亚基包含14个α螺旋和16个β链，折叠成一个小结构域和一个大结构域。辅酶结合赖氨酸残基位于一个亚基的大、小结构域之间以及一个结晶学相关亚基的大结构域之间的界面处。大的磷酸吡哆醛结合结构域的折叠和活性位点的位置与转氨酶中的相似。在转氨酶中参与磷酸吡哆醛结合的大多数残基在酪氨酸酚裂解酶的结构中是保守的。酪氨酸酚裂解酶的两个二聚体，每个二聚体都具有与天冬氨酸转氨酶中发现的类似的结构域结构，通过分子中心的疏水簇和相互缠绕的N端臂结合在一起。
