Reactivity of recombinant and mutant vanadium bromoperoxidase from the red alga Corallina officinalis.
Vanadium bromoperoxidase (VBPO) from the marine red alga Corallina officinalis has been cloned and heterologously expressed in Esherichia coli. The sequence for the full-length cDNA of VBPO from C. officinalis is reported. Steady state kinetic analyses of monochlorodimedone bromination reveals the recombinant enzyme behaves similarly to native VBPO from the alga. The kinetic parameters (K(m)(Br-)=1.2 mM, K(m)(H(2)O(2))=17.0 microM) at the optimal pH 6.5 for recombinant VBPO are similar to reported values for enzyme purified from the alga. The first site-directed mutagenesis experiment on VBPO is reported. Mutation of a conserved active site histidine residue to alanine (H480A) results in the loss of the ability to efficiently oxidize bromide, but retains the ability to oxidize iodide. Kinetic parameters (K(m)(I-)=33 mM, K(m)(H(2)O(2))=200 microM) for iodoperoxidase activity were determined for mutant H480A. The presence of conserved consensus sequences for the active sites of VBPO from marine sources shows its usefulness in obtaining recombinant forms of VBPO. Furthermore, mutagenesis of the conserved extra-histidine residue shows the importance of this residue in the oxidation of halides by hydrogen peroxide.
Carter
Jayme N
JN
Department of Chemistry and Biochemistry, University California Santa Barbara, Santa Barbara, CA 93106-9510, USA.
Beatty
Kimberly E
KE
Simpson
Matthew T
MT
Butler
Alison
A
D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000502:physiology; D006861:Hydrogen Peroxide / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000461:Rhodophyta / Q000201:enzymology*; D016415:Sequence Alignment; D014638:Vanadates / Q000378:metabolism
D020418:Algal Proteins; D011994:Recombinant Proteins; D014638:Vanadates; D006861:Hydrogen Peroxide; D010544:Peroxidases; C026195:bromide peroxidase