Reengineering the nucleotide cofactor specificity of the RecA protein by mutation of aspartic acid 100.
We have recently obtained evidence for a direct linkage between the S0.5 (S0.5 is the substrate concentration required for half-maximal velocity) value of a nucleoside triphosphate and the conformational state of the RecA-ssDNA complex, with an S0.5 value of 125 microM or less required for stabilization of the strand exchange-active conformation. For example, although ATP and ITP are hydrolyzed by the RecA protein with the same turnover number (18 min-1), ATP (S0.5 = 45 microM) functions as a cofactor for the strand exchange reaction, whereas ITP (S0.5 = 500 microM) is inactive as a strand exchange cofactor. The RecA protein crystal structure suggests that cofactor specificity is determined by Asp100, which likely forms a hydrogen bond with the exocyclic 6-amino group of ATP; the higher S0. 5 value for ITP is presumably due to unfavorable interactions between Asp100 and the 6-carbonyl group of the inosine ring. To test this hypothesis, we prepared a mutant RecA protein in which Asp100 was replaced by an asparagine residue. The S0.5(ITP) for the [D100N]RecA protein is 125 microM, indicating favorable interactions between the Asn100 side chain and the 6-carbonyl group of ITP. Correspondingly, ITP functions as a cofactor for the strand exchange activity of the [D100N]RecA protein. This result demonstrates the importance of the residue at position 100 in determining nucleotide cofactor specificity and underscores the importance of the S0.5 value in the RecA protein-promoted strand exchange reaction.
Stole
E
E
Department of Biochemistry, The Johns Hopkins University, School of Public Health, Baltimore, Maryland 21205, USA.
Bryant
F R
FR
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007293:Inosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D015202:Protein Engineering; D011938:Rec A Recombinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D017931:DNA Primers; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D007293:Inosine Triphosphate; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases