Molecular cloning, structure, and reactivity of the second bromoperoxidase from Ascophyllum nodosum

The sequence of bromoperoxidase II from the brown alga Ascophyllum nodosum was determined from a full length cloned cDNA, obtained from a tandem mass spectrometry RT-PCR-approach. The clone encodes a protein composed of 641 amino-acids, which provides a mature 67.4 kDa-bromoperoxidase II-protein (620 amino-acids). Based on 43% sequence homology with the previously characterized bromoperoxidase I from A. nodosum, a tertiary structure was modeled for the bromoperoxidase II. The structural model was refined on the basis of results from gel filtration and vanadate-binding studies, showing that the bromoperoxidase II is a hexameric metalloprotein, which binds 0.5 equivalents of vanadate as cofactor per 67.4 kDa-subunit, for catalyzing oxidation of bromide by hydrogen peroxide in a bi-bi-ping-pong mechanism (kcat = 153 s−1, 22 °C, pH 5.9). Bromide thereby is converted into a bromoelectrophile of reactivity similar to molecular bromine, based on competition kinetic data on phenol bromination and correlation analysis. Reactivity provided by the bromoperoxidase II mimics biosynthesis of methyl 4-bromopyrrole-2-carboxylate, a natural product isolated from the marine sponge Axinella tenuidigitata.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► The structure of bromoperoxidase II from the brown alga Ascophyllum nodosum was determined from a full length cloned cDNA. ► Bromoperoxidase II is a hexameric metalloprotein that binds per 67.4 kDa-subunit 0.5 equivalents of vanadate. ► Vanadate is the cofactor for catalyzing oxidation of bromide by hydrogen peroxide. ► Enzymatic bromide oxidation furnishes a bromoelectrophile showing similar reactivity to molecular bromine. ► Bromoperoxidase II mimics biosynthesis of marine natural product methyl 4-bromopyrrole-2-carboxylate.