Unveiling hidden catalytic contributions of the conserved His/Trp-III in tyrosine recombinases: assembly of a novel active site in Flp recombinase harboring alanine at this position.
The catalytic pentad of tyrosine recombinases, that assists the tyrosine nucleophile, includes a conserved histidine/tryptophan (His/Trp-III). Flp and Cre harbor tryptophan at this position; most of their kin recombinases display histidine. Contrary to the conservation rule, Flp(W330F) is a much stronger recombinase than Flp(W330H). The hydrophobicity of Trp330 or Phe330 is utilized in correctly positioning Tyr343 during the strand cleavage step of recombination. Why then is phenylalanine almost never encountered in the recombinase family at this conserved position? Using exogenous nucleophiles and synthetic methylphosphonate or 5'-thiolate substrates, we decipher that Trp330 also assists in the activation of the scissile phosphate and the departure of the 5'-hydroxyl leaving group. These two functions are consistent with the hydrogen bonding property of Trp330 as well as its location in structures of the Flp recombination complexes. However, van der Waals contact between Trp330 and Arg308 may also be important for the phosphate activation step. A structure based suppression strategy permits the inactive variant Flp(W330A) to be rescued by a second site mutation A339M. Modeling alanine and methionine at positions 330 and 339, respectively, in the Flp crystal structure suggests a plausible mechanism for active site restoration. Successful suppression suggests the possibility of evolving, by design, new active site configurations for tyrosine recombination.
Ma
Chien-Hui
CH
Molecular Genetics and Microbiology, University of Texas, Austin, 1 University Station A5000, Austin, TX 78712-0162, USA.
Kwiatek
Agnieszka
A
Bolusani
Swetha
S
Voziyanov
Yuri
Y
Jayaram
Makkuni
M
D000409:Alanine / Q000737:chemistry / Q000502:physiology*; D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000187:drug effects; D017124:Conserved Sequence / Q000502:physiology; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000502:physiology*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008957:Models, Genetic; D008958:Models, Molecular; D009154:Mutation; D045522:Recombinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014439:Tyramine / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism
D045522:Recombinases; D014443:Tyrosine; D006639:Histidine; D014364:Tryptophan; D006861:Hydrogen Peroxide; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D000409:Alanine; D014439:Tyramine