At the heart of vanadium haloperoxidase enzymes
Chemists of the Biocatalysis research group at the Van 't Hoff Institute for Molecular Sciences have obtained very fine details of the active centre of vanadium haloperoxidase enzymes. Their research elucidates the remarkable performance of both a chloroperoxidase they discovered twenty-odd years ago and a mutant they developed several years later. The enzymes have potential for application in antibacterial and antifungal agents.
In a recent publication in the Journal of the American Chemical Society (JACS) the HIMS chemists establish the role of protons in the active centre of the enzymes, which has been quite elusive up to now. They determined the protonation environment of the vanadate in close cooperation with experts in solid state NMR at the University of Delaware (USA).
Vanadium haloperoxidases are a class of enzymes, which are rather common in nature. For instance they help seaweeds in their defense against intruders. They also enable the pathology of certain fungi parasitizing on plants, by demolishing the cell wall of plant cells.
Their action is based upon the production of hypohalous acids through the reaction of halides, mainly chloride and bromide, with hydrogen peroxide. The fungi secrete chloroperoxidases that convert chloride (Cl-) into hypochlorous acid (HOCl), which is also the active ingredient in household bleach. In the seaweeds bromoperoxidases convert bromide (Br-) into hypobromous acid (HOBr), which has similar properties.
Because of their ability to form hypohalous acids the enzymes hold potential for a broad field of applications ranging from laundry detergents to antibacterial and antifungal agents. The HIMS Biocatalysis group has investigated vanadium bromo- and chloroperoxidases for almost thirty years lead by professor Ron Wever, who retired last year.
In the beginning of the nineties of the last century the group discovered a vanadium chloroperoxidase sufficiently stable for real application. This has led to many scientific publications as well as two patents, one of which describes the use of the enzyme as a bleaching and disinfecting component in household products such as laundry detergents. The other concerns the use in paints that prevent the attachment of algae and other organisms on ship's hulls and maritime constructions such as drilling rigs.
The latter application called for an optimisation of the enzyme function at a pH of 8, the pH of seawater. Though so-called directed evolution the group succeeded in developing a mutant enzyme, which displays a hundredfold increased activity at pH 8 compared with the native enzyme. Former group member Rokus Renirie, now working at IVAM UvA BV, a research and consultancy firm in the field of sustainability, performed part of this work. The antibacterial and antifungal application of the mutant enzyme is part of his portfolio at IVAM.
Next to the development of applications considerable research has been conducted on the factors determining the catalytic conversion rates of both the native and mutant enzymes. By using an array of spectroscopy methods such as X-ray, UV-VIS, EPR, V-EXAFS and V-NMR combined with steady-state and pre-steady state kinetics and mutagenesis studies detailed understanding of the enzyme structure and the conversion mechanism has been obtained.
It is known now that vanadium is located at the active centre of the enzyme in the form of vanadate (HVO42-). In a two-step mechanism first the hydrogen peroxide binds to the centre, followed by the reaction of a halide ion with this 'peroxo-intermediate' resulting in the formation of hypohalous acid (HOX).
The recent JACS publication, featuring Wever and Renirie as co-authors, provides further detailed insight. In close cooperation with the solid state NMR research group of professor Tatjana Polenova at the University of Delaware (USA) the researchers succeeded in establishing the protonation environment of the vanadate in the active centre.
Thanks to the so-called Magic Angle Spinning (MAS) method the solid state NMR reveals far more detail than regular NMR performed in solution. By combining the experimental results with quantum mechanical density functional theory (DFT) calculations it was possible to derive the protonation environment. It turned out this environment was not only pH dependent but also differed between the native and mutant enzyme, thus confirming that the pH optimum in enzyme activity was determined by the protonation environment.
The high sensitivity of the solid state NMR also led to an improvement of the data of the wild type enzyme as compared to a previous 2006 JACS publication. Finally the current research suggests that a water molecule is bound axially to the vanadate cofactor and dissociates during the reaction with hydrogen peroxide resulting in a stable peroxo intermediate.
Rupal Gupta, Guangjin Hou, Rokus Renirie, Ron Wever, Tatyana Polenova: 51V NMR Crystallography of Vanadium Chloroperoxidase and Its Directed Evolution P395D/L241V/T343A Mutant: Protonation Environments of the Active Site. J. Am. Chem. Soc., 2015, 137 (16), pp 5618–5628. DOI: 10.1021/jacs.5b02635