YjeQ, an essential, conserved, uncharacterized protein from Escherichia coli, is an unusual GTPase with circularly permuted G-motifs and marked burst kinetics

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 a]. (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 muM (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.