PMID:12220175
| Citation |
Daigle, DM, Rossi, L, Berghuis, AM, Aravind, L, Koonin, EV and Brown, ED (2002) YjeQ, an essential, conserved, uncharacterized protein from Escherichia coli, is an unusual GTPase with circularly permuted G-motifs and marked burst kinetics. Biochemistry 41:11109-17 |
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| Abstract |
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. |
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| Keywords |
Amino Acid Motifs; Amino Acid Sequence; Catalysis; Conserved Sequence; Escherichia coli/enzymology; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/isolation & purification; Escherichia coli Proteins/metabolism; GTP Phosphohydrolases/chemistry; GTP Phosphohydrolases/isolation & purification; GTP Phosphohydrolases/metabolism; Guanosine Triphosphate/metabolism; Heterotrimeric GTP-Binding Proteins/chemistry; Heterotrimeric GTP-Binding Proteins/isolation & purification; Heterotrimeric GTP-Binding Proteins/metabolism; Hydrolysis; Kinetics; Molecular Sequence Data; Peptide Fragments/isolation & purification; Protein Binding; Recombinant Proteins/isolation & purification; Sequence Homology, Amino Acid; Substrate Specificity |
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