Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/3/2011
Publication Date: 2/4/2011
Citation: Ullah, A.H., Sethumadhavan, K., Mullaney, E.J. 2011. Vanadate inhibition of fungal phyA and bacterial appA2 histidine acid phosphatases. Journal of Agricultural and Food Chemistry. (59)1739-1743. Interpretive Summary: Phytic acid, a storage form of phosphate, is a plant metabolite copiously found in the seeds of legumes such as soybean. The phosphates are organically bound to phytic acid; therefore, are unavailable to simple-stomached animals that lack an enzyme that degrades the metabolite. Fortunately, microbes produce enzymes that are capable of degrading phytic acid and thereby releasing phosphates that are known nutrients both for plants and animals. Phytic acid is also known to be anti-nutrients because it could bind important metals such as iron, manganese, copper, calcium, etc., and cause deficiencies in mineral nutrition. When phytase enzyme is supplemented to animal feed containing soybean and other phytic acid rich plant seeds, the animal could obtain phosphates from the meal and as an added benefit they are not subjected to mineral deficiencies. The molecular details of fungal phytase now revealed that they contain another functional site by virtue of which they could breakdown peroxides. However, the fungal phytase first have to be shut down by a chemical named vanadium oxide before it could breakdown peroxides. In this paper we have shown that very little amount of vanadium oxide, both ortho- and meta- forms, could shut down the phytase activity. The inhibitory concentration of vanadium oxide for both fungal and bacterial phytase is low enough to have a physiological significance. The phytase enzyme is a unique example of one biocatalyst fully capable of working as two biocatalysts. The molecular switch is however vanadium oxide. We have performed detailed studies of the inhibition kinetics of phytase by vanadium oxide to appreciate how this inorganic molecule could serve as a master switch to shut down one enzymatic activity and to turn on another enzymatic activity. This is quite an exciting development because phytases could be utilized to perform chloroperoxidase activity in the laboratory to synthesize drugs with utmost specificities.
Technical Abstract: The fungal PhyA protein, which was first identified as an acid optimum phosphomonoesterase (EC 18.104.22.168), could also serve as a vanadate haloperoxidase (EC 22.214.171.124) provided the acid phosphatase activity is shutdown by vanadate. To understand how vanadate inhibits both phytate and pNPP degrading activities of fungal PhyA phytase and bacterial AppA2 phytase, kinetic experiments were performed in the presence and absence of orthovanadate and metavanadate under various acidic pH. Orthovanadate was found to be a potent inhibitor at pH 2.5 to 3.0. A 50% activity of fungal phytase was inhibited at 0.56 µM by orthovanadate. However, metavanadate preferentially inhibited the bacterial AppA2 phytase (50% inhibition at 8 µM) over the fungal phytase (50% inhibition at 40 µM). While in bacterial phytase the Km was not affected by ortho- or metavanadate, the Vmax was reduced. In fungal phytase, both the Km and Vmax was lowered. The vanadate exists as anion at pH 3.0 and possibly binds to the active center of phytases that has cluster of positively charged Arg, Lys, and His residues below the enzymes’ isoelectric point (pI). The active site fold of haloperoxidase was shown to be very similar to fungal phytase. The vanadate anions binding to cationic residues in the active site at acidic pH thus serves as a molecular switch to turn off phytase activity while turning on the haloperoxidase activity. The fungal PhyA phytase’s active site housing two distinct reactive center one for phosphomonoesterase and the other for haloperoxidase is a unique example of how one protein could catalyze two dissimilar reactions controlled by vanadate.