Sankaranarayanan Ramasamy, Cherney Maia M, Cherney Leonid T, Garen Craig R, Moradian Fatemeh, James Michael N G
Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
J Mol Biol. 2008 Jan 25;375(4):1052-63. doi: 10.1016/j.jmb.2007.11.025. Epub 2007 Nov 19.
Mycobacterium tuberculosis ornithine carbamoyltransferase (Mtb OTC) catalyzes the sixth step in arginine biosynthesis; it produces citrulline from carbamoyl phosphate (CP) and ornithine (ORN). Here, we report the crystal structures of Mtb OTC in orthorhombic (form I) and hexagonal (form II) space groups. The molecules in form II are complexed with CP and l-norvaline (NVA); the latter is a competitive inhibitor of OTC. The asymmetric unit in form I contains a pseudo hexamer with 32 point group symmetry. The CP and NVA in form II induce a remarkable conformational change in the 80s and the 240s loops with the displacement of these loops towards the active site. The displacement of these loops is strikingly different from that seen in other OTC structures. In addition, the ligands induce a domain closure of 4.4 degrees in form II. Sequence comparison of active-site residues of Mtb OTC with several other OTCs of known structure reveals that they are virtually identical. The interactions involving the active-site residues of Mtb OTC with CP and NVA and a modeling study of ORN in the form II structure strongly rule out an earlier proposed mechanistic role of Cys264 in catalysis and suggest a possible mechanism for OTC. Our results strongly support the view that ORN with an already deprotonated N(epsilon) atom is the species that binds to the enzyme and that one of the phosphate oxygen atoms of CP is likely to be involved in accepting a proton from the doubly protonated N(epsilon) atom of ORN. We have interpreted this deprotonation as part of the collapse of the transition state of the reaction.
结核分枝杆菌鸟氨酸氨甲酰基转移酶（Mtb OTC）催化精氨酸生物合成的第六步；它从氨甲酰磷酸（CP）和鸟氨酸（ORN）生成瓜氨酸。在此，我们报告了Mtb OTC在正交晶系（I型）和六方晶系（II型）空间群中的晶体结构。II型分子与CP和L - 正缬氨酸（NVA）复合；后者是OTC的竞争性抑制剂。I型的不对称单元包含一个具有32点群对称性的假六聚体。II型中的CP和NVA诱导80s和240s环发生显著构象变化，这些环向活性位点位移。这些环的位移与其他OTC结构中的情况显著不同。此外，配体在II型中诱导了4.4度的结构域闭合。将Mtb OTC活性位点残基与其他几种已知结构的OTC进行序列比较，发现它们几乎相同。Mtb OTC活性位点残基与CP和NVA之间的相互作用以及对II型结构中ORN的建模研究有力地排除了先前提出的Cys264在催化中的机制作用，并提出了一种可能的OTC机制。我们的结果有力地支持了这样一种观点，即具有已经去质子化的N(ε)原子的ORN是与酶结合的物种，并且CP的一个磷酸氧原子可能参与从ORN的双质子化N(ε)原子接受一个质子。我们将这种去质子化解释为反应过渡态崩溃的一部分。
