Structural Insights into Regioselectivity in the Enzymatic Chlorination of Tryptophan

Abstract

The regioselectively controlled introduction of chlorine into organic molecules is an important biological and chemical process. This importance derives from the observation that many pharmaceutically active natural products contain a chlorine atom. Flavin-dependent halogenases are one of the principal enzyme families responsible for regioselective halogenation of natural products. Structural studies of two flavin-dependent tryptophan 7-halogenases (PrnA and RebH) have generated important insights into the chemical mechanism of halogenation by this enzyme family. These proteins comprise two modules: a flavin adenine dinucleotide (FAD)-binding module and a tryptophan-binding module. Although the 7-halogenase studies advance a hypothesis for regioselectivity, this has never been experimentally demonstrated. PyrH is a tryptophan 5-halogenase that catalyzes halogenation on tryptophan C5 position. We report the crystal structure of a tryptophan 5-halogenase (PyrH) bound to tryptophan and FAD. The FAD-binding module is essentially unchanged relative to PrnA (and RebH), and PyrH would appear to generate the same reactive species from Cl(-), O(2), and 1,5-dihydroflavin adenine dinucleotide. We report additional mutagenesis data that extend our mechanistic understanding of this process, in particular highlighting a strap region that regulates FAD binding, and may allow communication between the two modules. PyrH has a significantly different tryptophan-binding module. The data show that PyrH binds tryptophan and presents the C5 atom to the reactive chlorinating species, shielding other potential reactive sites. We have mutated residues identified by structural analysis as recognizing the tryptophan in order to confirm their role. This work establishes the method by which flavin-dependent tryptophan halogenases regioselectively control chlorine addition to tryptophan. This method would seem to be general across the superfamily.

Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase–thioesterase enzyme pair

“…6 A and B) are superimposable. Although we could not determine a substrate pyrrolyl-S-ACP cocrystal structure for either Bmp2 or Mpy16, the position of the pyrrole binding sites for Bmp2 and Myp16 was inferred by a structural alignment of Bmp2 and Mpy16 with the crystal structures of substrate-bound forms of the flavin-dependent tryptophan-7-chlorinases PrnA (16) and RebH (17). Fittingly, the postulated pyrrole-binding site in Bmp2 and Mpy16 (Fig.…”

“…Notably, the active site of Bmp2-TM exhibited no perturbation of the bound FAD cofactor with respect to WT Bmp2, nor did it lead to any apparent change in halogen binding properties of the enzyme, as demonstrated by conservation of its specificity for bromide. Already, for flavin-dependent tryptophan halogenases, it has been demonstrated that amino acid side chains that constitute the substrate binding site control the regiochemical outcomes for halogen additions (17,24,25 this observation to flavin-dependent halogenases that catalyze halogenation of aromatic substrates acylated to ACPs in demonstrating that side chains of residues lining the putative halogenase active site play a role in controlling substrate access, and potentially in specifying the positions on the pyrrole ring that are accessible to halogenation. Unfortunately, in the case of Mpy16, efforts to alter the putative substrate-binding cavity to resemble that of Bmp2 resulted in insolubility of the mutant Mpy16 enzymes.…”

The Ternary Complex of PrnB (the Second Enzyme in the Pyrrolnitrin Biosynthesis Pathway), Tryptophan, and Cyanide Yields New Mechanistic Insights into the Indolamine Dioxygenase Superfamily

“…The first enzyme, PrnA (tryptophan 7-halogenase), incorporates the chlorine into the substrate tryptophan (6). Structural and biochemical analyses of both tryptophan 7 and 5-halogenase (7)(8)(9)(10) have established a novel chemical mechanism of hypohalous acid formation at the flavin cofactor, followed by N-chlorolysine formation (9) and finally regioselective halogenation of tryptophan (controlled by orientation of substrate) (10,11). PrnB converts 7-Cl-tryptophan into monodechloroamino-pyrrolnitrin, although the chirality of the PrnB substrate has remained unclear (4).…”

A Fivefold Parallelized Biosynthetic Process Secures Chlorination of Armillaria mellea (Honey Mushroom) Toxins

“…In contrast, our current study focuses on flavin-dependent halogenases, which employ FAD as a cofactor and use a halide anion and molecular oxygen to produce hypochlorous acid as a halogenating agent. Protein crystallography greatly helped in understanding the structural basis of regioselective formation of the carbon-halogen bond (50)(51)(52)(53). A. mellea halogenase genes encode the signature motif GW(A/V)W(F/L)I.…”