Daigle Denis M, Rossi Laura, Berghuis Albert M, Aravind L, Koonin Eugene V, Brown Eric D
Antimicrobial Research Centre, Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada L8N 3Z5.
Biochemistry. 2002 Sep 17;41(37):11109-17. doi: 10.1021/bi020355q.
The Escherichia coli protein YjeQ represents a protein family whose members are broadly conserved in bacteria and have been shown to be indispensable to the growth of E. coli and Bacillus subtilis [Arigoni, F., et al. (1998) Nat. Biotechnol. 16, 851]. Proteins of the YjeQ family contain all sequence motifs typical of the vast class of P-loop-containing GTPases, but show a circular permutation, with a G4-G1-G3 pattern of motifs as opposed to the regular G1-G3-G4 pattern seen in most GTPases. All YjeQ family proteins display a unique domain architecture, which includes a predicted N-terminal OB-fold RNA-binding domain, the central permuted GTPase module, and a zinc knuckle-like C-terminal cysteine cluster. This domain architecture suggests a possible role for YjeQ as a regulator of translation. YjeQ was overexpressed, purified to homogeneity, and shown to contain 0.6 equiv of GDP. Steady state kinetic analyses indicated slow GTP hydrolysis, with a k(cat) of 9.4 h(-)(1) and a K(m) for GTP of 120 microM (k(cat)/K(m) = 21.7 M(-)(1) s(-)(1)). YjeQ also hydrolyzed other nucleoside triphosphates and deoxynucleotide triphosphates such as ATP, ITP, and CTP with specificity constants (k(cat)/K(m)) ranging from 0.2 to 1.0 M(-)(1) s(-)(1). Pre-steady state kinetic analysis of YjeQ revealed a burst of nucleotide hydrolysis for GTP described by a first-order rate constant of 100 s(-)(1) as compared to a burst rate of 0.2 s(-)(1) for ATP. In addition, a variant in the G1 motif of YjeQ (S221A) was substantially impaired for GTP hydrolysis (0.3 s(-)(1)) with a less significant impact on the steady state rate (1.8 h(-)(1)). In summary, E. coli YjeQ is an unusual, circularly permuted P-loop-containing GTPase, which catalyzes GTP hydrolysis at a rate 45 000 times greater than that of turnover.
大肠杆菌蛋白YjeQ代表一个蛋白家族，其成员在细菌中广泛保守，并且已被证明对大肠杆菌和枯草芽孢杆菌的生长不可或缺[Arigoni, F., 等人 (1998) 《自然生物技术》16, 851]。YjeQ家族的蛋白包含了大量含P环的GTP酶典型的所有序列基序，但呈现出一种环形排列，其基序模式为G4-G1-G3，与大多数GTP酶中常见的规则G1-G3-G4模式相反。所有YjeQ家族蛋白都展示出一种独特的结构域架构，其中包括一个预测的N端OB折叠RNA结合结构域、中央的重排GTP酶模块以及一个锌指样C端半胱氨酸簇。这种结构域架构表明YjeQ可能作为翻译调节因子发挥作用。YjeQ被过量表达并纯化至同质，结果显示其含有0.6当量的GDP。稳态动力学分析表明其GTP水解缓慢，催化常数k(cat)为9.4 h⁻¹，对GTP的米氏常数K(m)为120 μM（k(cat)/K(m) = 21.7 M⁻¹ s⁻¹）。YjeQ还能水解其他核苷三磷酸和脱氧核苷三磷酸，如ATP、ITP和CTP，其特异性常数（k(cat)/K(m)）范围为0.2至1.0 M⁻¹ s⁻¹。对YjeQ的预稳态动力学分析显示，GTP的核苷酸水解有一个爆发期，其一级速率常数为100 s⁻¹，而ATP的爆发速率为0.2 s⁻¹。此外，YjeQ的G1基序中的一个变体（S221A）的GTP水解能力大幅受损（0.3 s⁻¹），对稳态速率的影响较小（1.8 h⁻¹）。总之，大肠杆菌YjeQ是一种不同寻常的、环形排列的含P环GTP酶，其催化GTP水解的速率比周转速率快45000倍。
