The Vanadium Iodoperoxidase from the Marine Flavobacteriaceae Species Zobellia galactanivorans Reveals Novel Molecular and Evolutionary Features of Halide Specificity in the Vanadium Haloperoxidase Enzyme Family

Abstract

Vanadium haloperoxidases (VHPO) are key enzymes that oxidize halides and are involved in the biosynthesis of organo-halogens. Until now, only chloroperoxidases (VCPO) and bromoperoxidases (VBPO) have been characterized structurally, mainly from eukaryotic species. Three putative VHPO genes were predicted in the genome of the flavobacterium Zobellia galactanivorans, a marine bacterium associated with macroalgae. In a phylogenetic analysis, these putative bacterial VHPO were closely related to other VHPO from diverse bacterial phyla but clustered independently from eukaryotic algal VBPO and fungal VCPO. Two of these bacterial VHPO, heterogeneously produced in Escherichia coli, were found to be strictly specific for iodide oxidation. The crystal structure of one of these vanadium-dependent iodoperoxidases, Zg-VIPO1, was solved by multiwavelength anomalous diffraction at 1.8 Å, revealing a monomeric structure mainly folded into ␣-helices. This three-dimensional structure is relatively similar to those of VCPO of the fungus Curvularia inaequalis and of Streptomyces sp. and is superimposable onto the dimeric structure of algal VBPO. Surprisingly, the vanadate binding site of Zg-VIPO1 is strictly conserved with the fungal VCPO active site. Using site-directed mutagenesis, we showed that specific amino acids and the associated hydrogen bonding network around the vanadate center are essential for the catalytic properties and also the iodide specificity of Zg-VIPO1. Altogether, phylogeny and structure-function data support the finding that iodoperoxidase activities evolved independently in bacterial and algal lineages, and this sheds light on the evolution of the VHPO enzyme family. H alogenated compounds have various biological functions in nature, ranging from chemical defense to signaling. Indeed, halogenation (i.e., iodination, bromination, or chlorination) is an efficient strategy used to increase the biological activity of secondary metabolites and involves many different halogenating enzymes (1-4). Among them, vanadium-dependent haloperoxidases (VHPO) contain the bare metal oxide vanadate as a prosthetic group. In the presence of hydrogen peroxide, VHPO enzymes catalyze the oxidation of halides according to the reaction H 2 O 2 ϩ X Ϫ ϩ H ϩ ¡ H 2 O ϩ HOX, wherein X Ϫ represents a halide ion and may be Cl Ϫ , Br Ϫ , or I Ϫ (4). A variety of halocarbons can subsequently be generated if the appropriate nucleophilic acceptors are present. The nomenclature of vanadium-dependent haloperoxidases is based on the most electronegative halide they can oxidize: chloroperoxidases (VCPO) can catalyze the oxidation of three different halides, i.e., chloride, bromide and iodide; bromoperoxidases (VBPO) can oxidize only bromide and iodide; and iodoperoxidases (VIPO) are specific for iodide.The first VBPO was discovered 30 years ago, in the brown alga Ascophyllum nodosum (5). Since then, structural and mechanistic studies have focused on two types of eukaryotic VHPO, namely, VCPO from the pathogenic fungus Curvularia inaequalis (6) a...

Vanadium haloperoxidases: From the discovery 30 years ago to X-ray crystallographic and V K-edge absorption spectroscopic studies

“…This alignment bears the two following short domains highly conserved in all VHPO proteins and related to vanadate binding (see below). A c c e p t e d M a n u s c r i p t 10 bacterial nonspecific acid phosphatases as outgroup [7,56]. In this phylogenetic tree ( Figure 1) the VHPOs from red and brown algae group together with some VBPOs from Cyanobacteria, suggesting a common origin of all algal VHPOs.…”

“…In this phylogenetic tree ( Figure 1) the VHPOs from red and brown algae group together with some VBPOs from Cyanobacteria, suggesting a common origin of all algal VHPOs. Inside the brown algal VHPO sub-group, two branches appear, one including the first VBPO of A. nodosum and the VIPO from L. digitata and the other bearing the second isoform of A. nodosum and the VBPO of L. digitata [56]. The red algal VBPOs form a strong monophyletic group with few cyanobacterial VHPO homologous proteins, one of which has recently been characterized as VBPO in a Synechococcus strain [91].…”

“…No specific iodinated substrate has been yet identified in L. digitata, but this particular VHPO may be involved in the very efficient accumulation of iodide in external tissues of L. digitata [14,74,75]. Another VIPO was recently characterized at the structural level in the marine flavobacterium Z. galactanivorans, which belongs to the bacteroidetes and is associated with both red and brown seaweeds [56].…”

“…Indeed, algal VHPOs display several additional insertions, forming helices and beta-strands that are not present in the fungal or bacterial VHPOs [56]. The comparison of all the known haloperoxidase structures shows that a fungal or bacterial VHPO monomer can be superimposed onto the dimer of an algal VHPO, with the N-terminal helix-bundle 1 of the single peptide chain in the monomeric forms being replaced by the 'back-side' of the Cterminal helix-bundle belonging to the second chain of the homodimeric form ( Figure 2C).…”

“…specific for iodide oxidation, in brown algae of the Laminariaceae [55]. Using this assay, novel VIPO were recently characterized in the marine bacteria Zobellia galactanivorans [56].…”