The Fluorinase from Streptomyces cattleya Is Also a Chlorinase

Abstract

Keywordschlorinase; enzyme catalysis; fluorinase; halogenation; S-adenosylmethionine Streptomyces cattleya is unusual in that it elaborates organofluorine metabolites and in particular secretes fluoroacetate and 4-fluorothreonine as secondary metabolites when incubated in a medium supplemented with fluoride ions. [1,2] The first committed step on the biosynthetic pathway to these fluorometabolites involves the enzymatic mediated synthesis of 5′-fluoro-5′-deoxyadenosine (5′-FDA) from S-adenosyl-L-methionine (SAM) and fluoride ions. [3][4][5] After depurination, 5′-FDA is metabolized first to 5-fluoro-5-deoxyribose-1-phosphate (5′-FRP) [6] and then to fluorocetaldehyde. [7] Fluoroacetaldehyde is converted separately into fluoroacetate [8] and 4-fluorothreonine [9] (Scheme 1).The fluorination enzyme, which has been the subject of a recent structural study, [10] has been isolated and fully characterized, the gene has been cloned, and the enzyme has been overexpressed. A stereochemical study that involved (5′S)-[5′-2 H]-SAM has indicated that the C-F bond of 5′-FDA replaces the C-S bond of SAM with an inversion of configuration. [11,12] This observation, as well as a theoretical study [13] and structural analysis [10] all suggest an S N 2 reaction mechanism for the fluorinase. Despite the high bonddissociation energy of the C-F bond (the strongest bond in organic chemistry [14] ), we herein report that the fluorinase operates in the reverse direction and that it utilizes a chloride ion. Incubation of 5′-FDA with L-( 13 C-methyl)methionine resulted in the formation of 13 Cmethyl-SAM, which was identified by HPLC-ES-MS. A similar experiment with Lselenomethionine (L-Se-met) and 5′-FDA resulted in a sixfold more-efficient reaction to generate L-Se-SAM, which was readily identified through the mass-spectrometry fragmentation product of methylselenoadenosine (MeSeAdo) by its signature isotope fingerprint in HPLC-ES-MS. Comparison with the V max values that were calculated at saturating kinetics (assays run in each direction) indicated that the equilibrium lies in favor of 5′-FDA by a factor of three (see Scheme 2).Efforts were made to replace the fluoride ion with a chloride ion as a substrate for the fluorinase by using standard assay protocols. Incubation with chloride ions in the presence ** We thank the BBSRC for funding (H.D. and D.A.R.) and GSK and the Pro-Bio-Faraday for a Studentship (R.P.M.).

Structural Perspective on Enzymatic Halogenation

“…In a unique reversal of the typical methylation reaction catalyzed by AdoMet-dependent enzymes, 5′-FDAS exploits l -Met as an excellent leaving group for an S N 2-type reaction. 3,38 The fluorinase catalyzes attack of F − on AdoMet to produce 5′-fluoro-5′-deoxyadenosine (5′-FDA) (Scheme 1E). 5′-FDAS can also catalyze chlorination, although the reverse reaction (dechlorination) is thermodynamically favored.…”

“…5′-FDAS can also catalyze chlorination, although the reverse reaction (dechlorination) is thermodynamically favored. 38 Recently, a homologue of 5′-FDAS, SalL from the marine bacterium Salinispora tropica , was characterized and shown to catalyze nucleophilic chlorination under native conditions. 39 SalL, which has 35% sequence identity with 5′-FDAS, converts AdoMet to 5′-chloro-5′-deoxyadenosine (5′-ClDA) and cannot perform fluorination.…”

Halomethane Biosynthesis: Structure of a SAM‐Dependent Halide Methyltransferase from Arabidopsis thaliana

“…This enzyme is chemically similar and almost structurally identical to the fluorinase, an enzyme from Streptomyces cattleya that utilizes both fluoride and to a lesser extent chloride ion in a similar nucleophilic attack to C5' carbon of SAM. [22][23][24] X-ray structural data is in place for the nucleophilic fluorinase [23] and chlorinase [21] enzymes, as well as representatives of almost all of the other known halogenases. [15] Therefore, the Scheme 1.…”

AdoMet analog synthesis and utilization: current state of the art

“…SalL and FDAS catalyze the reversible formation of l -Met and 5′-chloro or 5’-fluoro-5′-deoxyadenosine (ClDA or FDA, respectively) from AdoMet and chloride or fluoride, respectively, where the equilibrium typically favors the reactants (Fig. 2e) [41–45]. In this pioneering work [19], SalL and FDAS were found to catalyze the production of six differentially S -alkylated AdoMet analogs from their respective l -Met analogs and commercially available ClDA or FDA.…”