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J. Biol. Chem., Vol. 277, Issue 21, 18849-18859, May 24, 2002
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From the The Bacillus subtilis endospore coat
protein CotA shows laccase activity. By using comparative modeling
techniques, we were able to derive a model for CotA based on the known
x-ray structures of zucchini ascorbate oxidase and Cuprinus
cereneus laccase. This model of CotA contains all the structural
features of a laccase, including the reactive surface-exposed copper
center (T1) and two buried copper centers (T2 and T3). Single amino
acid substitutions in the CotA T1 copper center (H497A, or
M502L) did not prevent assembly of the mutant proteins into the coat
and did not alter the pattern of extractable coat polypeptides.
However, in contrast to a wild type strain, both mutants produced
unpigmented colonies and spores unable to oxidize syringaldazine (SGZ)
and 2'2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The
CotA protein was purified to homogeneity from an overproducing
Escherichia coli strain. The purified CotA shows an
absorbance and a EPR spectra typical of blue multicopper oxidases. Optimal enzymatic activity was found at
Molecular and Biochemical Characterization of a Highly Stable
Bacterial Laccase That Occurs as a Structural Component of the
Bacillus subtilis Endospore Coat*
§,,,,,
Instituto de Tecnologia Química e
Biológica, Universidade Nova de Lisboa, Av. da República,
2781-901 Oeiras, Portugal, § Universidade Lusófona de
Humanidades e Tecnologias, Departamento de Engenharias e Tecnologias,
Av. do Campo Grande, 376, 1749-024 Lisboa, Portugal, and
¶ Department of Biology, Emory University,
Atlanta, Georgia 30322
pH 3.0 and at pH 7.0 for ABTS
or SGZ oxidation, respectively. The apparent Km values for ABTS and SGZ at 37 °C were of 106 ± 11 and 26 ± 2 µM, respectively, with corresponding
kcat values of 16.8 ± 0.8 and 3.7 ± 0.1 s
1. Maximal enzyme activity was observed at 75 °C
with ABTS as substrate. Remarkably, the coat-associated or the purified
enzyme showed a half-life of inactivation at 80 °C of about 4 and
2 h, respectively, indicating that CotA is intrinsically highly thermostable.
*
This work was supported by Grants from the Instituto de
Tecnologia Química e Biológica (ITQB) (to A. O. H.) and
POCTI/BME/32789/99 (to C. M. S.) and Fundação para a
Ciência e a Tecnologia Grant SFRH/BD/1167/2000 (to T. C.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
351-214469521; Fax: 351-214411277; E-mail: aoh@itqb.unl.pt.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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