Tryptophan 7-Halogenase (PrnA) Structure Suggests a Mechanism for Regioselective Chlorination

Abstract

Chlorinated natural products include vancomycin and cryptophycin A. Their biosyntheses involves regioselective chlorination by flavin-dependent halogenases. We report the structural characterization of tryptophan 7-halogenase (PrnA), which regioselectively chlorinates tryptophan. Tryptophan and FAD are separated by a 10Å-long tunnel and bound by distinct enzyme modules. The FAD module is conserved in halogenases and is related to flavin-dependent monooxygenases. Based on biochemical studies, crystal structures and by analogy with monooxygenases, we predict FADH 2 reacts with O 2 making peroxy-flavin which is decomposed by Cl −. The resulting HOCl is guided through the tunnel, to tryptophan, where it is activated to participate in electrophilic aromatic substitution. In addition to man-made chemicals, there are nearly four thousand chlorinated and brominated natural products (1), including drugs such as vancomycin (2), rebeccamycin (3) and cryptophycin A (4). The de-novo chemical synthesis of complex natural products is often too expensive or too difficult to be practical. Their production relies on fermentation and introducing diversity in such molecules requires protein engineering. This has been hampered by a lack of understanding of the molecular basis of the biological regioselective halogenation mechanism. Metal-dependent haloperoxidases were once thought to catalyze all halogenation reactions in biology and fall into two classes. Both, heme-iron-dependent enzymes (5) and vanadium-dependent enzymes (6), have been structurally characterized. Although different in structure, both form a metal-bound hydrogen peroxide, which reacts with halide ions producing a metal-bound hypohalite ion. This ion dissociates from the metal as hypohalous acid (5,6) where, in solution, it reacts with substrate. Such halogenation lacks regioselectivity and substrate specificity (7). Perhydrolases are now recognized not to be involved in halometabolite biosynthesis (8). A new halogenase was reported by Dairi et al. identifying the gene for the chlorinating enzyme in chlorotetracycline biosynthesis (9). The gene product showed no similarity to haloperoxidases. Studies of the antifungal compound pyrrolnitrin from Pseudomonas fluorescens identified two related genes (prnA and prnC), coding for two halogenating enzymes (10) (fig. S1). Both contain a flavin binding site (9-13) and exhibit weak sequence homology to flavin-dependent monooxygenase enzymes (14). PrnA catalyzes the regioselective chlorination of the 7-position of tryptophan (15). Turnover requires that FAD is reduced to FADH 2 (by flavin reductase) and that O 2 is present (11). One sentence: The crystal structure of tryptophan 7-halogenase indicates that the halogenation of important metabolites in bacteria proceeds by the novel use of hypohalous acid to achieve regioselective substitution.

Chlorine-35/37 NMR Spectroscopy of Solid Amino Acid Hydrochlorides: Refinement of Hydrogen-Bonded Proton Positions Using Experiment and Theory

“…Interactions between tryptophan moieties and chloride ions are of direct biological importance as illustrated, for example, by a recent publication which suggests a mechanism for the electrophilic addition of chloride to tryptophan by the PrnA enzyme. 16 Furthermore, interactions between chloride and various amino acid sidechains are responsible for chloride ion selectivity and transport in various ion channels including the ClC type. [17][18][19][20] The present study provides new insights into how the local chloride binding environment is manifested in the chlorine CS tensor.…”

Direct Investigation of Covalently Bound Chlorine in Organic Compounds by Solid‐State 35Cl NMR Spectroscopy and Exact Spectral Line‐Shape Simulations

“…[5] Recent studies show that covalently bound chlorine is also important in biological chemistry where, for example, the tryptophan 7-halogenase was found to selectively chlorinate tryptophan moieties. [6] Herein, we show that with the combination of an ultrahigh magnetic field (B 0 = 21.1 T) and the state-of-the-art WURST-QCPMG pulse sequence, [7] it is possible to acquire highquality 35 Cl NMR spectra of organic compounds that contain a covalently bound chlorine atom in powder samples in a reasonable amount of time. We have acquired 35 Cl NMR spectra of 5-chlorouracil (1); the pesticide 2-chloroacetamide (2); sodium chloroacetate (3); a,a'-dichloro-o-xylene (4); chlorothiazide (5), a diuretic pharmaceutical also known as diuril; 2,4'-dichloroacetophenone (6); and p-chlorophenylalanine (7), a chlorinated amino acid, which is used as an inhibitor of tryptophan hydroxylase.…”

RadH: A Versatile Halogenase for Integration into Synthetic Pathways

“…The kinetic parameters for RadH with selected substrates were determined (Table 1 and Figures S4–S10). Notably, the k cat values for RadH with iso‐quinoline 6 and coumarin 8 were significantly higher than those reported for Trp‐Hal enzymes with the natural substrate 3a, 13. Whilst kinetic analysis shows that monocillin II ( 1 ) is turned over more slowly than the other substrates, k cat / K m was not determined for 1 since saturation of the Michaelis–Menten curve was not attained.…”