Probing the active site of YjeE: a vital Escherichia coli protein of unknown function.
In the study described here, we have taken steps to characterize the YjeE protein, an Escherichia coli protein of unknown function that is essential for bacterial viability. YjeE represents a protein family whose members are broadly conserved in bacteria, absent from eukaryotes and contain both Walker A and B motifs, characteristic of P-loop ATPases. We have revisited the dispensability of the yjeE gene in E. coli and describe efforts to probe the function of the YjeE protein with in vitro biochemistry. We have looked critically for ATPase activity in the recombinant E. coli protein and have made vigilant use of site-directed variants in the Walker A [K41A (Lys41-->Ala) and T42A] and putative Walker B (D80Q) motifs. We noted that any hydrolysis of ATP by the wild-type E. coli protein might be attributed to background ATPase, since it was not appreciably different from that of the variants. To overcome potential contaminants, we turned to crystalline pure YjeE protein from Haemophilus influenzae that was found to hydrolyse ATP at a slow rate (kcat=1 h(-1)). We have also shown high-affinity binding to YjeE by ADP using equilibrium dialysis (K(d)=32 microM) and by fluorescence resonance energy transfer from a conserved tryptophan in YjeE to a fluorescent derivative of ADP, 2'-/3'-O-(N-methylanthraniloyl)adenosine 5'-O-diphosphate (K(d)=8 microM). Walker motif variants were notably impaired for ADP binding and T42A and D80Q mutations in yjeE were incapable of complementing the yjeE deletion strain.
Allali-Hassani
Abdellah
A
Department of Biochemistry and Antimicrobial Research Centre, McMaster University, 1200 Main Street, West Hamilton, ON, Canada L8N 3Z5.
Campbell
Tracey L
TL
Ho
Andy
A
Schertzer
Jeffrey W
JW
Brown
Eric D
ED
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D003956:Dialysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005816:Genetic Complementation Test; D014644:Genetic Variation / Q000235:genetics; D006193:Haemophilus influenzae / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D017384:Sequence Deletion / Q000235:genetics; D013816:Thermodynamics
D029968:Escherichia coli Proteins; C500230:YjeE protein, E coli; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases