Pillai Bindu, Cherney Maia M, Diaper Christopher M, Sutherland Andrew, Blanchard John S, Vederas John C, James Michael N G
Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, AB, Canada T6G 2H7.
Proc Natl Acad Sci U S A. 2006 Jun 6;103(23):8668-73. doi: 10.1073/pnas.0602537103. Epub 2006 May 24.
D-amino acids are much less common than their L-isomers but are widely distributed in most organisms. Many D-amino acids, including those necessary for bacterial cell wall formation, are synthesized from the corresponding L-isomers by alpha-amino acid racemases. The important class of pyridoxal phosphate-independent racemases function by an unusual mechanism whose details have been poorly understood. It has been proposed that the stereoinversion involves two active-site cysteine residues acting in concert as a base (thiolate) and an acid (thiol). Although crystallographic structures of several such enzymes are available, with the exception of the recent structures of glutamate racemase from Bacillus subtilis and of proline racemase from Trypanosoma cruzi, the structures either are of inactive forms (e.g., disulfide) or do not allow unambiguous modeling of the substrates in the active sites. Here, we present the crystal structures of diaminopimelate (DAP) epimerase from Haemophilus influenzae with two different isomers of the irreversible inhibitor and substrate mimic aziridino-DAP at 1.35- and 1.70-A resolution. These structures permit a detailed description of this pyridoxal 5'-phosphate-independent amino acid racemase active site and delineate the electrostatic interactions that control the exquisite substrate selectivity of DAP epimerase. Moreover, the active site shows how deprotonation of the substrates' nonacidic hydrogen at the alpha-carbon (pKa approximately 29) by a seemingly weakly basic cysteine residue (pKa approximately 8-10) is facilitated by interactions with two buried alpha-helices. Bacterial racemases, including glutamate racemase and DAP epimerase, are potential targets for the development of new agents effective against organisms resistant to conventional antibiotics.
D-氨基酸比其L-异构体少见得多，但在大多数生物体中广泛分布。许多D-氨基酸，包括细菌细胞壁形成所必需的那些，是由α-氨基酸消旋酶从相应的L-异构体合成的。重要的一类不依赖磷酸吡哆醛的消旋酶通过一种不寻常的机制起作用，其细节了解甚少。有人提出，立体反转涉及两个活性位点的半胱氨酸残基协同作用，一个作为碱（硫醇盐），另一个作为酸（硫醇）。尽管有几种这样的酶的晶体结构，但除了最近来自枯草芽孢杆菌的谷氨酸消旋酶和来自克氏锥虫的脯氨酸消旋酶的结构外，这些结构要么是无活性形式（例如二硫键），要么不允许在活性位点对底物进行明确建模。在这里，我们展示了流感嗜血杆菌中二氨基庚二酸（DAP）差向异构酶与不可逆抑制剂和底物模拟物氮杂环丙烷-DAP的两种不同异构体的晶体结构，分辨率分别为1.35 Å和1.70 Å。这些结构允许对这种不依赖磷酸吡哆醛5'-磷酸的氨基酸消旋酶活性位点进行详细描述，并描绘了控制DAP差向异构酶精细底物选择性的静电相互作用。此外，活性位点展示了一个看似弱碱性的半胱氨酸残基（pKa约为8 - 10）如何通过与两个埋藏的α-螺旋的相互作用促进底物α-碳上非酸性氢（pKa约为29）的去质子化。包括谷氨酸消旋酶和DAP差向异构酶在内的细菌消旋酶是开发有效对抗对传统抗生素耐药的生物体的新药物的潜在靶点。
