Structural Basis of Stereospecific Vanadium-Dependent Haloperoxidase Family Enzymes in Napyradiomycin Biosynthesis

Abstract

Vanadium-dependent haloperoxidases (VHPOs) from Streptomyces bacteria differ from their counterparts in fungi, macroalgae, and other bacteria by catalyzing organohalogenating reactions with strict regiochemical and stereochemical control. While this group of enzymes collectively uses hydrogen peroxide to oxidize halides for incorporation into electron-rich organic molecules, the mechanism for the controlled transfer of highly reactive chloronium ions in the biosynthesis of napyradiomycin and merochlorin antibiotics sets the Streptomyces vanadium-dependent chloroperoxidases apart. Here we report high-resolution crystal structures of two homologous VHPO family members associated with napyradiomycin biosynthesis, NapH1 and NapH3, that catalyze distinctive chemical reactions in the construction of meroterpenoid natural products. The structures, combined with site-directed mutagenesis and intact protein mass spectrometry studies, afforded a mechanistic model for the asymmetric alkene and arene chlorination reactions catalyzed by NapH1 and the isomerase activity catalyzed by NapH3. A key lysine residue in NapH1 situated between the coordinated vanadate and the putative substrate binding pocket was shown to be essential for catalysis. This observation suggested the involvement of the ε-NH2, possibly through formation of a transient chloramine, as the chlorinating species much as proposed in structurally distinct flavin-dependent halogenases. Unexpectedly, NapH3 is modified post-translationally by phosphorylation of an active site His (τ-pHis) consistent with its repurposed halogenation-independent, α-hydroxyketone isomerase activity. These structural studies deepen our understanding of the mechanistic underpinnings of VHPO enzymes and their evolution as enantioselective biocatalysts.

How Is Substrate Halogenation Triggered by the Vanadium Haloperoxidase from Curvularia inaequalis?

“…Halogenases and haloperoxidases use various mechanisms to transfer a halide (Cl – , Br – , I – ) to a substrate, namely, through electrophilic, nucleophilic, or radical-type pathways. − Understanding the enzymatic process of inserting a halide atom into an aliphatic or aromatic C–H bond is a crucial step in the biocatalytic application of such enzymes in the biotechnology industry and would make an environmentally benign alternative to the use of toxic metals and solvents . Bioengineering approaches to produce novel halogenated scaffolds would enable an alternative and sustainable synthesis route for fine chemicals and drugs, and hence extensive research has aimed to understand the reaction mechanisms and substrate scope of halogenases and haloperoxidases. ,, The frequent stereo- and regioselectivities of halogenases and haloperoxidases offer the possibility of high product yields at fast turnover rates in more environmentally sustainable conditions. − …”

Class II terpene cyclases: structures, mechanisms, and engineering

“…33 To date, class I TCs have been more extensively characterized with over 30 structures; only twelve class II TC structures are reported. 1,30,33,[44][45][46][47][48] In this section, we will review these twelve class II TCs with determined structures (Table 1).…”

“…9,21,126 To add further complexity, noncanonical TCs that are distinct in their sequences, structures, mechanisms, and cellular locations (i.e., soluble vs. membranebound) have been identied and characterized. 46,81,168 Research on class II TCs has spanned almost three decades since the initial structural determination of SHC. However, the community is only in its infancy in understanding how to successfully and logically engineer these enzymes to alter the products.…”

“…7B). 46 As a homodimer, each subunit of NapH1 contains both N- and C-terminal helical bundles with an interconnecting region; a vanadate-binding site is located in each subunit. In NapH1, the vanadate cofactor is stabilized by the dipole interaction from helix 12, forming charge–charge interactions with K372, R379, and R488.…”

Asymmetric catalysis by flavin‐dependent halogenases

“…6 Of these enzymes, only members of the latter two classes exhibit catalyst-controlled site-selectivity to enable precise placement of halogen substituents in diverse substrates. While tryptophan FDHs often possess some degree of specificity for L-tryptophan over D-tryptophan, [7][8][9][10] vanadium haloperoxidases and nonheme iron halogenases can catalyze stereoselective halogenation via olefin halocyclization 11,12 and aliphatic C-H functionalization, 13,14 respectively.…”