Angewandte
Chemie
binding site. The altered position of the
halogen does not, however, prevent the
enzyme from catalyzing the reaction.
In the second conformation (not
shown) the chlorine rotated out of the
halogen binding site into the “empty” l-
methionine sulfur binding site. Of course
in the catalytic situation, l-methionine
will occupy the space above the ribose
ring and there will be no possibility of
chlorine adopting this second orienta-
tion.
This study reveals that the fluorinase
will catalyze the combination of chloride
ions and SAM to generate 5’-ClDA. The
reaction is only observed in coupled-
enzyme assays that prevent the reverse
reaction from occurring. No organohal-
ogen products were detected when BrÀ
and IÀ were used in these reactions. This
reaction extends the repertoire of the
fluorinase and reveals a novel enzymatic
Figure 1. HPLC profile monitoring the fluorinase mediated transhalogenation reaction of 5’-
ClDA to 5’-FDA with time and using l-Se-methionine as a co-catalyst. l-Se-SAM accumulates as
a minor product.
chlorination reaction which operates by nucleophilic substi-
tution rather than through the electrophilic hypochlorite[17] or
flavin dependent enzymes[18] or by a recently reported
enzymatic chlorine radical process.[19] A combination of
nucleophilic chloride ion and SAM is responsible for the
production of chloromethane in fungi.[20,21] This is the only
other enzymatic reaction we are aware of in which ClÀ acts as
a nucleophile, interestingly with a different regiochemistry on
the same substrate.
Figure 2. Structure of the 5’-ClDA-fluorinase co-complex with contacts
and distances between the halogens and Ser-156 N and O atoms
shown. For 5’-ClDA, C=gray, N=blue, O=red, and Cl=purple. The
protein is similarly colored except C=yellow. Shown superimposed
over 5’-ClDA is 5’-FDA with C=green, F=orange, and N=blue. As
can be seen, the Cl atom lies further away from the backbone amide of
Ser-156.
Experimental Section
HPLC and NMR spectroscopy time course: The reaction mixtures for
HPLC were incubated at 378C with 5’-ClDA (0.3 mm), l-Se-
methionine (0.08 mm), KF (75m m), and fluorinase (6 mgmLÀ1
,
500 mL) in a final volume of 650 mL. Samples for HPLC analysis
(60 mL) were boiled at 958C (3 min) and the precipitated protein was
then removed by centrifugation. An aliquot (20 mL) of the clear
supernatant was used for HPLC analysis (Vivian 9012 UV/Vis
detector at 260 nm and 9050 solvent delivery module).
there are some small differences owing to crystal packing.
Only one subunit of the trimer is discussed.
Samples for 19F NMR (500 MHz) analysis were incubated at 378C
with 5’-FDA (0.5m m), L-methionine (40 mm), D2O (10%) and
fluorinase (6 mgmLÀ1, 5 00mL) in a final volume of 700 mL.
Preparations of ESI-MS sample: A sample (250 mL) for ESI-MS
analysis was incubated at 378C overnight with 5’-FDA (0.4 mm),
methionine (l-methionine-13C-methyl or selenomethionine; 15m m)
and fluorinase (6 mgmLÀ1) and then were boiled at 958C (3 min) and
the precipitated protein was then removed by centrifugation. The
supernatant was injected directly into the ESI-MS (MicroMass,
Manchester, UK) with MeCN as the solvent. The SAM analogue
molecules were detected in positive ion mode. Se-SAM underwent
fragmentation to 5’-methylselenoadenosine during ESI-MS analyses
(see the Supporting Information).
À
The difference electron density shows that the C5’ Cl
bond adopts two orientations that locate the organochlorine
atom in one of two positions. One conformer (shown in
Figure 2) is similar to that observed for 5’-FDA when bound
to the fluorinase, however, the chlorine atom is displaced by
1.3 relative to the location of the fluorine in the 5’-FDA
complex. The chlorine atom is anchored to the main chain
amide of Ser-156 by a hydrogen bond and a polar contact to
the side chain hydroxy group of the same residue. Similar
noncovalent contacts are also observed between the fluorine
atom and Ser-156 although the distances are shorter for the 5’-
FDA structure. We attribute this change in position to be a
consequence of the larger van der Waals radius of the Cl atom
relative to the F atom. The protein underwent no significant
change in response to the larger atom, rather the substrate
adjusts its position which perhaps suggests a rigid ligand
Crystallization of the 5-ClDA co-complex: FDAS (4 mgmLÀ1
)
was incubated overnight with 5’-ClDA (20 mm) at room temperature.
The solution was then crystallized by vapor diffusion against a
reservoir containing PEG 1000 (32%), phosphate-citrate buffer
solution (0.1m, pH 4.2), and Li2SO4 (0.2m). A single crystal was
selected and flash cooled to 100 K in a nitrogen stream. A data set was
recorded at the European synchrotron radiation facility by using the
Angew. Chem. Int. Ed. 2006, 45, 759 –762
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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