SummaryThe serine integrase, Int, from the Streptomyces phage fC31 mediates the integration and excision of the phage genome into and out of the host chromosome. Integrases usually require a recombination directionality factor (RDF) or Xis to control integration and excision and, as fC31 Int only mediates integration in the absence of other phage proteins, we sought to identify a fC31 RDF. Here we report that the fC31 early protein, gp3 activated attL x attR recombination and inhibited attP x attB recombination. Gp3 binds to Int in solution and when Int is bound to the attachment sites. Kinetic analysis of the excision reaction suggested that gp3 modifies the interactions between Int and the substrates to form an active recombinase. In the presence of gp3, Int assembles an excision synaptic complex and the accumulation of the integration complex is inhibited. The structure of the excision synaptic complex, like that of the hyperactive mutant of Int, IntE449K, appeared to be biased towards one that favours the production of correctly joined products. The functional properties of fC31 gp3 resemble those of the evolutionarily unrelated RDF from phage Bxb1, suggesting that these two RDFs have arisen through convergent evolution.

Most temperate phages encode an integrase for integration and excision of the prophage. Integrases belong either to the lambda Int family of tyrosine recombinases or to a subgroup of the serine recombinases, the large serine recombinases. Integration by purified serine integrases occurs efficiently in vitro in the presence of their cognate (~50 bp) phage and host attachment sites, attP and attB respectively. Serine integrases require an accessory protein, Xis, to promote excision, a reaction in which the products of the integration reaction, attL and attR, recombine to regenerate attP and attB. Unlike other directional recombinases, serine integrases are not controlled by proteins occupying accessory DNA-binding sites. Instead, it is thought that different integrase conformations, induced by binding to the DNA substrates, control protein-protein interactions, which in turn determine whether recombination proceeds. The present review brings together the evidence for this model derived from the studies on phiC31 integrase, Bxb1 integrase and other related proteins.

Choices choices: The fluorinase enzyme from Streptomyces cattleya (catalyzes the formation of a CF bond from fluoride ions) also has the capacity to utilize a chloride ion although it has a clear preference for the fluoride ion. The enzyme mediates a nucleophilic chlorination reaction, which is an unusual mechanism for enzymatic chlorination.

The integrase (Int) from phage ϕC31 acts on the phage and host-attachment sites, attP and attB, to form an integrated prophage flanked by attL and attR. Excision (attL × attR recombination) is prevented, in the absence of accessory factors, by a putative coiled-coil motif in the C-terminal domain (CTD). Int has a serine recombinase N-terminal domain, required for synapsis of recombination substrates and catalysis. We show here that the coiled-coil motif mediates protein–protein interactions between CTDs, but only when bound to DNA. Although the histidine-tagged CTD (hCTD) was monomeric in solution, hCTD bound cooperatively to three of the recombination substrates (attB, attL and attR). Furthermore, when provided with attP and attB, hCTD brought these substrates together in a synaptic complex. Substitutions in the coiled-coil motif that greatly reduce Int integration activity, L460P and Y475H, prevented CTD–CTD interactions and led to defective DNA binding and no detectable DNA synapsis. A substitution, E449K, in full length Int confers the ability to perform excision in addition to integration as it has gained the ability to synapse attL × attR. hCTDE449K was similar to hCTD in DNA binding but unable to form the CTD synapse suggesting that the CTD synapse is not essential but could be part of the mechanism that controls directionality.

Recently a fluorination enzyme was identified and isolated from Streptomyces cattleya, as the first committed step on the metabolic pathway to the fluorinated metabolites, fluoroacetate and 4-fluorothreonine. This enzyme, 5′-fluoro-5′-deoxy adenosine synthetase (FDAS), has been shown to catalyze C-F bond formation by nucleophilic attack of fluoride ion to S-adenosyl-L-methionine (SAM) with the concomitant displacement of L-methionine to generate 5′-fluoro-5′-deoxy adenosine (5′-FDA). Although the structures of FDAS bound to both SAM and products have been solved, the molecular mechanism remained to be elucidated. We now report site directed mutagenesis studies, structural analyses and isothermal calorimetry (ITC) experiments. The data establish the key residues required for catalysis and the order of substrate binding. Fluoride ion is not readily distinguished from water by protein X-ray crystallography, however using chloride ion (also a substrate) with mutants of low activity has enabled the halide ion to be located in nonproductive co-complexes with SAH and SAM. The kinetic data suggest the positively charged sulfur of SAM is a key requirement in stabilizing the transition state. We propose a molecular mechanism for FDAS in which fluoride weakly associates with the enzyme exchanging two water molecules for protein ligation. The binding of SAM expels remaining water associated with fluoride ion and traps the ion in a pocket positioned to react with SAM, generating L-methionine and 5′-FDA. L-SAM then dissociates from the enzyme followed by 5′-FDA.