Department of Chemistry, University of Georgia, Athens, Georgia 30602, United States.
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, United States.
Biochemistry. 2021 Jan 26;60(3):231-244. doi: 10.1021/acs.biochem.0c00635. Epub 2021 Jan 11.
We have examined the reaction of serovar typhimurium tryptophan (Trp) synthase αβ complex with l-Trp, d-Trp, oxindolyl-l-alanine (OIA), and dioxindolyl-l-alanine (DOA) in the presence of disodium (dl)-α-glycerol phosphate (GP), using stopped-flow spectrophotometry and X-ray crystallography. All structures contained the d-isomer of GP bound at the α-active site. (3)-OIA reacts with the pyridoxal-5'-phosphate (PLP) of Trp synthase to form a mixture of external aldimine and quinonoid complexes. The α-carboxylate of OIA rotates about 90° to become planar with the PLP when the quinonoid complex is formed, resulting in a conformational change in the loop of residues 110-115. The COMM domain of the Trp synthase-OIA complex is found as a mixture of two conformations. The (3)-diastereomer of DOA binds about 5-fold more tightly than (3)-OIA and also forms a mixture of aldimine and quinonoid complexes. DOA forms an additional H-bond between the 3-OH of DOA and βLys-87. l-Trp does not form a covalent complex with the PLP of Trp synthase. However, d-Trp forms a mixture of two external aldimine complexes which differ in the orientation of the α-carboxylate. In one conformation, the α-carboxylate is in the plane of the PLP, while in the other conformation, the α-carboxylate is perpendicular to the PLP plane. These results confirm that the stereochemistry of the transient indolenine quinonoid intermediate in the mechanism of Trp synthase is (3) and demonstrate the linkage between aldimine and quinonoid reaction intermediates in the β-active site and allosteric communications with the α-active site.
我们研究了鼠伤寒沙门氏菌色氨酸（Trp）合酶 αβ 复合物与 l-Trp、d-Trp、吲哚基-l-丙氨酸（OIA）和二吲哚基-l-丙氨酸（DOA）在二钠盐（dl）-α-甘油磷酸（GP）存在下的反应，采用停流分光光度法和 X 射线晶体学。所有结构都包含结合在 α 活性部位的 GP d-异构体。（3）-OIA 与色氨酸合酶的吡哆醛-5'-磷酸（PLP）反应形成外部希夫碱和醌型复合物的混合物。当形成醌型复合物时，OIA 的α-羧基旋转约 90°变得与 PLP 共面，导致残基 110-115 环的构象变化。色氨酸合酶-OIA 复合物的 COMM 结构域被发现为两种构象的混合物。（3）-DOA 的对映异构体比（3）-OIA 结合得更紧密约 5 倍，也形成希夫碱和醌型复合物的混合物。DOA 在 DOA 的 3-OH 和βLys-87 之间形成额外的 H 键。l-Trp 不与色氨酸合酶的 PLP 形成共价复合物。然而，d-Trp 形成两种外部希夫碱复合物的混合物，其区别在于α-羧酸盐的取向。在一种构象中，α-羧酸盐位于 PLP 平面内，而在另一种构象中，α-羧酸盐垂直于 PLP 平面。这些结果证实了色氨酸合酶机制中瞬态吲哚啉醌型中间物的立体化学为（3），并证明了β-活性部位中希夫碱和醌型反应中间物之间的联系以及与α-活性部位的变构通讯。
