Athappilly F K, Hendrickson W A
Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA.
Structure. 1995 Dec 15;3(12):1407-19. doi: 10.1016/s0969-2126(01)00277-5.
Acetyl-coenzyme A carboxylase catalyzes the first committed step of fatty acid biosynthesis. Universally, this reaction involves three functional components all related to a carboxybiotinyl intermediate. A biotinyl domain shuttles its covalently attached biotin prosthetic group between the active sites of a biotin carboxylase and a carboxyl transferase. In Escherichia coli, the three components reside in separate subunits: a biotinyl domain is the functional portion of one of these, biotin carboxy carrier protein (BCCP).
We have expressed natural and selenomethionyl (Se-met) BCCP from E. coli as biotinylated recombinant proteins, proteolyzed them with subtilisin Carlsberg to produce the biotinyl domains BCCP and Se-met BCCPsc, determined the crystal structure of Se-met BCCPsc using a modified version of the multiwavelength anomalous diffraction (MAD) phasing protocol, and refined the structure for the natural BCCPsc at 1.8 A resolution. The structure may be described as a capped beta sandwich with quasi-dyad symmetry. Each half contains a characteristic hammerhead motif. The biotinylated lysin is located at a hairpin beta turn which connects the two symmetric halves of the molecule, and its biotinyl group interacts with a non-symmetric protrusion from the core.
This first crystal structure of a biotinyl domain helps to unravel the central role of such domains in reactions catalyzed by biotin-dependent carboxylases. The hammerhead structure observed twice in BCCPsc may be regarded as the basic structural motif of biotinyl and lipoyl domains of a superfamily of enzymes. The new MAD phasing techniques developed in the course of determining this structure enhance the power of the MAD method.
乙酰辅酶A羧化酶催化脂肪酸生物合成的第一步关键反应。一般来说，该反应涉及与羧基生物素中间体相关的三个功能组分。一个生物素结构域在生物素羧化酶和羧基转移酶的活性位点之间穿梭其共价连接的生物素辅基。在大肠杆菌中，这三个组分存在于不同的亚基中：生物素结构域是其中之一生物素羧基载体蛋白（BCCP）的功能部分。
我们将来自大肠杆菌的天然和硒代甲硫氨酸（Se-met）BCCP表达为生物素化重组蛋白，用卡尔伯格枯草杆菌蛋白酶对其进行蛋白水解以产生生物素结构域BCCP和Se-met BCCPsc，使用多波长反常衍射（MAD）相位测定方案的改进版本确定Se-met BCCPsc的晶体结构，并在分辨率为1.8 Å的条件下对天然BCCPsc的结构进行精修。该结构可描述为具有准二元对称性的带帽β三明治结构。每一半都包含一个特征性的锤头基序。生物素化的赖氨酸位于连接分子两个对称半部的发夹β转角处，其生物素基团与核心的不对称突出物相互作用。
生物素结构域的首个晶体结构有助于揭示此类结构域在生物素依赖性羧化酶催化反应中的核心作用。在BCCPsc中观察到两次的锤头结构可被视为一类酶超家族的生物素和硫辛酰结构域的基本结构基序。在确定该结构过程中开发的新MAD相位测定技术增强了MAD方法的能力。
