FUCUS VANADIUM PEROXIDASE: MINIMUM CATALYTIC DOMAIN SIZE RETAINING PEROXIDASE ACTIVITY

Vanadium peroxidase catalyzes the extracellular assembly of Fucus zygote cell surface adhesive (Vreeland & Epstein 1996, Modern Meth. Plant Anal. 17, 95–116). Our goal is to identify the catalytic, self‐associating and wall targeting functional domains of algal vanadium peroxidase to understand its role in algal propagule adhesion. As a first step, we truncated our recombinant Fucus vanadium peroxidase (GenBank AF053411) for catalytic domain identification. Recombinant constructs were prepared which reduced the C‐terminal catalytic domain at either or both N‐ and C‐terminal ends. Recombinant proteins were expressed in E. coli , refolded from cytoplasm and inclusion bodies and tested for vanadium‐specific o‐dianisidine peroxidase activity. Preliminary results demonstrated peroxidase activity when the 40 kDa catalytic domain was truncated on both ends to 24 kDa. Further terminal and internal truncation is needed to fully define the minimal catalytic unit, which could be as small as 15–20 kDa within the 73 kDa monomer. The very small catalytic unit in Fucus vanadium peroxidase is not unexpected considering the rigid bundled helical vanadate frame in the Curvularia fungal vanadium peroxidase (Macedo‐Ribeiro et al. 1999, J. Biol. Inorg. Chem. 4, 209–219). We conclude that interactions between the N‐terminal noncatalytic domain and the C‐terminal catalytic domain, found in the crystalline Ascophyllum enzyme (Weyland et al. 1999, J. Mol. Biol. 293, 595–611), are unnecessary for peroxidase activity. Other conserved amino acids in the C‐terminal half of Fucus vanadium peroxidase, peripheral to the helical core, could participate in protein surface functions such self‐association and wall targeting.