Study On The Fluorinating Enzyme From The Marine-Derived Bacterium Streptomyces Xinghaiensis NRRL B24674

Fluorinase was first identified from the bacterium Streptomyces cattleya,an enzyme which catalyses the combination of S-adenosyl-L-methionine(SAM)and a fluoride ion to generate 5'-fluorodeoxy adenosine(5'-FDA)and L-methionine through a SN2 nucleophilic substitution reaction with a fluoride ion as the nucleophile to attack the C5' of SAM.The Streptomyces xinghaiensis NRRL B-24674 was the first marine-derived bacterium to generate a fluorinated natural product---fluoroacetate.We verified the existence of fluorinase from Streptomyces xinghaiensis NRRL B24674 in vitro and discussed the characteristics.The main contents and results include:(1)Gene sequence of the fluorinase was deduced from the whole genome of Streptomyces xinghaiensis NRRL B24674 and codon-optimised to be cloned into a pET-28a(+)vector.The plasmid pET-28a(+)flA was transformed into E.coli BL21(DE3)for expression.When over-expression was incuded with 0.01 mM isopropylthiogalactoside(IPTG)at 16?,the soluble protein was ideal.The overlap PCR method was used to introduce pET-28a(+)-D16E,pET-28a(+)-Y77A,pET-28a(+)-T80A,pET-28a(+)-T80S,pET-28a(+)-F156A and pET-28a(+)-S158A mutants.Then all mutants were induced for over-expression and purification.(2)The putatiuve fluorinating enzyme purified in the experiment was able to generate 5'-FDA,which was detected/momitored/comfirmed by HPLC and LC-MS.This prove/confirmed the existence of fluorinase in Streptomyces xinghaiensis NRRL B24674.Intriguingly,this enzyme was rather active in an acidic environment with a pH value as low as 4.With a basic condition(pH 8-9.5),its activity drops significantly.The optimal temperature for S.xinghaiensis fuorianse is around 40? as expected.It was possible a metal ion dependent enzyme.Cu2+ and Zn2+ can considerably inhibit activity.Kinetics assay(Km=7.04±0.94?M,Kcat=0.277±0.007 min-1,Kcat/Km = 39.5±1.51 mM-1.min-1)indicated the fluorinase from S.xinghaiensis was the most efficient one compared with other fluorinases from different sources reported previously.Thermal shift assays indicated the wild-type enzyme was more stable than all mutants.50 mM Tris-HCl buffer(pH 7.0)was better to maintain the stability and the biological activity.The chlorinase reaction indicated the fluorinated enzyme purified in the experiment also had chlorinase activity.(3)HPLC and CD measurements of wild-type and S158A mutant indicated that S158 was a key residue for hydrogen bonding with fuorine ions.D16 was crucial for substrate SAM binding and the F156 residue provided the rear of the hydrophobic pocket which is necessary to assist desolvation of fuoride ions.D16E and 156A mutants are detrimental to enzyme activity.T80A and Y77A severely abolished activity in our case,consistent with the mechanism that T80 and Y77 were able to facilitate halide binding.