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.

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The transcript marker PCA3 is a powerful predictor of primary PCa but the inclusion of EZH2, prostein, and TRPM8 adds even more to the diagnostic power. The finding of a significantly higher mRNA expression of three different genes (prostein, PSA, TRPM8) in organ-confined tumors compared to non-organ-confined tumors as well as the multi-marker PCa prediction model developed in the retrospective model system on prostatectomies could be of clinical importance for diagnostic purposes, and should be verified in diagnostic biopsies.

Dedicated to Prof. Franz Lingens on the occasion of his 80 th birthday.Abstract: Tryptophan 7-halogenase (Trp 7-hal) catalyses the regioselective chlorination and bromination of tryptophan. For halogenating activity, Trp 7-hal requires FADH 2 produced from FAD and NADH by a flavin reductase, halide ions (chloride or bromide), molecular oxygen and tryptophan as the organic substrate. Investigations of the flavin dependence showed that purified Trp 7-hal itself does not contain flavin. Keeping the Trp 7-hal separated from the flavin reductase during the reaction revealed that Trp 7-hal can use diffusible FADH 2 produced by a flavin reductase showing that direct contact between the halogenase and the flavin reductase is not absolutely necessary. Thus, the reaction also proceeds when chemically reduced flavin is used. For the catalytic regeneration of FADH 2 , the organometallic complex (pentamethylcyclopentadienyl)rhodium-bipyridine {[Cp*Rh(bpy)(H 2 O)] 2 þ } can be employed as the redox catalyst with formate as the electron donor. With this chemoenzymatic system about 85% yields of the product formed by the two-component enzyme system consisting of Trp 7-hal and a flavin reductase were obtained.