C O M M U N I C A T I O N S
FAD(C4a)-OOH toward productive chlorination (via generation of
HOCl, which is proposed to affect N-chlorination of an active-site
lysine side chain to form lysine chloramine as the proximal
halogenating species)9c versus competing autoxidation to FAD.
Whereas KtzQ is analogous to all of the previously characterized
tryptophan halogenases in its prefence for unmodified L-Trp, KtzR
is novel in that it has a ∼120 fold prefence for 7-Cl-L-Trp over
L-Trp. The substrate binding site in KtzQ is well conserved with
PrnA, RebH, ThaL, and PyrH and all of the amino acids responsible
for binding tryptophan are present. In contrast, two aromatic
residues which stack against the indole ring of tryptophan in the
PrnA and RebH structures, H101 and W455 in PrnA and H109
and W466 in RebH, are replaced with glutamine and leucine
residues respectively in KtzR (Table S1). Structural analysis is
warranted to determine if and then how these changes in the active
site residues allow KtzR to differentiate 7-Cl-L-Trp from L-Trp.
In summary, our initial characterization of the three enzymes
KtzQRS make it likely that the regioselective double halogenation
in kutzneride biosynthesis occurs on free L-Trp by sequential action
of the KtzQR pair. Kinetic characterization shows KtzQ acts first
to chlorinate at the 7-position of L-Trp and then KtzR installs the
second chlorine at the 6-position of 7-Cl-L-Trp to generate 6,7-
diCl-L-Trp. Thus, while KtzD, Q, and R are now characterized
halogenases, the chlorination of the piperazate moiety has not yet
been accounted for and may require further genomic analysis of
the Kutzneria producer.
Figure 2. HPLC analysis of the KtzQ and KtzR catalyzed halogenations:
(a) KtzQ incubated with L-Trp; (b) KtzQ incubated with 6-Cl-L-Trp; (c)
KtzR incubated with L-Trp; (d) KtzR incubated with 7-Cl- L-Trp; (e) KtzQ
and KtzR incubated with L-Trp.
product identity was also confirmed by ESI-MS ([M - H]- m/z )
270.9, 272.9, and 274.9) (Figure S6). Comparison of 1H NMR data
of the product and authentic 6,7-diCl-L-Trp confirmed the site of
chlorination at position 6 of the indole ring (Figure S7). Remarkably,
the catalytic efficiency for halogenation of position 6 of 7-Cl-L-
Trp was better than for L-Trp itself. The kcat ratio for 7-Cl-L-Trp/
L-Trp as substrate (1.4 min-1/0.08 min-1) favors 7-Cl-L-Trp by
∼18-fold, and the Km value for 7-Cl-L-Trp at 114 µM is ∼7-fold
lower than for L-Trp at 808 µM, thus the presence of a chloride at
the 7-position of the indole ring results in a ∼120-fold increase in
KtzR efficiency relative to umodified L-Trp. KtzR, like KtzQ, was
not active for introduction of chloride to piperazic acid or γ,δ-
dehydropiperazic acid.
Acknowledgment. We thank Prof. Robert S. Phillips for pro-
viding the authentic 7-chloro-L-Tryptophan used in these studies.
We also thank Elizabeth S. Sattely for technical assistance and
careful proofreading of the manuscript. This work was supported
by an NIH grant to C.T.W. (GM20011) and by an NIH NRSA
fellowship to J.R.H. (1 F32 GM083464-01).
Supporting Information Available: Figures S1-S7, Table S1,
detailed experimental procedures and spectral data. This material is
Tandem incubations of L-Trp with KtzQ and KtzR along with
the flavin reductase KtzS or SsuE and NADH and FAD yielded
the 6,7-diCl-L-Trp product (Figure 2e). From these results and the
individual enzyme kinetics, we conclude that the tandemly orga-
nized ktzQ and ktzR genes encode two FADH2-dependent haloge-
nases that work sequentially on free L-Trp in the order of KtzQ
followed by KtzR to form 6,7-diCl-L-Trp (Figure 1B). It is then
likely that 6,7-diCl-L-Trp is the monomer incorporated into the
growing kutzneride assembly line by the KtzH adenylation domain.
The final conversion to the diClPIC moiety in mature kutzneride
is postulated to be achieved by epoxidation of the indole 2,3-double
bond by the hemeprotein KtzM with intramolecular capture of the
epoxide by the amide nitrogen.2
KtzQ is a regiospecific tryptophan-7-halogenase analogous to
the enzymes RebH8 and PrnA9 that act in the first steps of re-
beccamycin and pyrrolnitrin biosynthesis, respectively. The regi-
oselectivity for introduction of chlorine at the 6-position of the
indole ring of L-Trp and 7-Cl-L-Trp renders KtzR a homologue of
ThaL6 in the thienodolin pathway. ThaL has a turnover of 2.8 min-1
while the Trp-7-halogenases PrnA and RebH perform at 0.1 to 1.4
chlorinations per minute; these values are within range of those
measured for KtzQ and KtzR. The kcat values may be faster in vivo
if enzyme complexes exist to channel the FADH2 and resultant
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