Chapter 11
Downloaded by UNIV OF OKLAHOMA on October 26, 2014 | http://pubs.acs.org Publication Date: August 30, 2007 | doi: 10.1021/bk-2007-0974.ch011
Structural Studies of Vanadium Haloperoxidases: Insight into Halide Specificity, Stability, and Enzyme Mechanism Jennifer Littlechild, Esther Garcia-Rodriguez, Elizabeth Coupe, Aaron Watts, and Mikail Isupov Henry Wellcome Building for Biocatalysis, School of Biosciences, University of Exeter, Exeter, United Kingdom
Crystallographic studies of the vanadium haloperoxidase found in Corallina red algae has revealed details of the structure of these enzymes which has increased our understanding of halide specificity, stability, substrate binding and enzymatic mechanism. A n efficient process to produce the enzyme in recombinant form after refolding of inclusion bodies has been developed. A novel truncated mutant dimeric form of the enzyme has been constructed for use in commercial biotransformation experiments.
Corallina officinalis is a red seaweed from the rhodophyta family, which also includes Chondrus and Porphyra. The red coloration is due to the presence of the pigment phycoerythrin that reflects red light and absorbs blue. This pigment allows the algae to photosynthesise at greater depths because blue light penetrates water to a greater depth than light of longer wavelengths. C. officinalis grows to about 5 inches in length and can be distinguished from other rhodophyta by the symmetrical branching of the thalli. C officinalis can be seen throughout the North Western and North Eastern Atlantic including in rock pools in Ladram Bay, Devon, U K where it is seen growing approximately 1-5 inches below the water surface. A related C. pilulifera species is found off the coast of Japan. The Corallina species have been shown to contain a bromoperoxidase that both binds and is dependent on vanadate for its activity. This enzyme is of particular industrial interest due to its known stability in organic solvents and its ability to withstand a wide pH range and temperatures up to 80°C (1-3). 136
© 2007 American Chemical Society
In Vanadium: The Versatile Metal; Kustin, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.
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Structure of the Corallina bromoperoxidases The structure of C officinalis vanadium haloperoxidase was solved to 2.3A in 2000 by Isupov et ai, (4) and showed a 595 amino acid chain folded into a single a + p type domain. The structure does not contain any disulfide bridges as found in the related Ascophyllum bromoperoxidase (5) although there are two cysteines present, and no post-translational modifications were observed. There are twelve monomers, each consisting of 19 a-helices which are 6 to 26 residues in length, eight 3, helices and 14 p-strands which are mainly involved in phairpins. One surface of the monomer is flat and upon dimerisation this surface forms the central region resulting in two four-helical bundles at the centre of each dimer. The active site cleft uses residues from both monomers, with the residues of one predominantly being responsible for the bottom of the active site binding the vanadate, while the other constitutes the top region of the cleft . The dimers then interact to form a dodecamer with a 23 cubic point symmetry, which is approximately 150A in diameter (Figure 1). The N-terminal helices point away from the main structure and form a central cavity in the dodecamer (Figure 2) which has no known function Two vanadate atoms are associated with each dimeric subunit (4) and are co-ordinated in a trigonal bipyramidal geometry with hydroxide and His496 in axial positions and three non-protein (solvent) oxygen atoms in equatorial positions. The original structure was solved with phosphate in place of the vanadium due to the high concentration of phosphate in the crystallisation conditions. Phosphate will prevent vanadate binding and it was observed that i f phosphate binds to vanadium haloperoxidases phosphatase activity can be observed (
