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Applied and Environmental Microbiology, September 1998, p. 3290-3299, Vol. 64, No. 9
Institute for
Microbiology1 and
Institute for Organic
Chemistry and Isotope Research,2
University of Stuttgart, D-70550 Stuttgart, and National
Research Center for Biotechnology, D-38124
Braunschweig,3 Germany, and
Centre
for Biotechnology,
Received 17 April 1998/Accepted 25 June 1998
Muconate cycloisomerases play a crucial role in the
bacterial degradation of aromatic compounds by converting
cis,cis-muconate, the product of catechol ring cleavage, to
(4S)-muconolactone. Chloromuconate
cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate
cycloisomerases from Pseudomonas putida
PRS2000 and Acinetobacter "calcoaceticus" ADP1. We show that they transform, in addition to
cis,cis-muconate, 3-fluoro-, 2-methyl-, and
3-methyl-cis,cis-muconate with high specificity constants
but not 2-fluoro-, 2-chloro-, 3-chloro-, or
2,4-dichloro-cis,cis-muconate. Based on known
three-dimensional structures, variants of P. putida
muconate cycloisomerase were constructed by
site-directed mutagenesis to contain amino acids found in equivalent
positions in chloromuconate cycloisomerases. Some of
the variants had significantly increased specificity constants for
3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and
22-fold increases, respectively, for the former substrate). These
kinetic improvements were not accompanied by a change from protoanemonin to cis,cis-dienelactone as the product of
3-chloro-cis,cis-muconate conversion. The rate of
2-chloro-cis,cis-muconate turnover was not significantly
improved, nor was this compound dehalogenated to any significant
extent. However, the direction of 2-chloro-cis,cis-muconate cycloisomerization could be influenced by amino acid exchange. While
the wild-type enzyme discriminated only slightly between the two
possible cycloisomerization directions, some of the enzyme variants
showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that
can be changed independently of each other.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Substrate Specificity of and Product Formation by
Muconate Cycloisomerases: an Analysis of Wild-Type Enzymes and
Engineered Variants
*
Corresponding author. Mailing address: Institute for
Microbiology, University of Stuttgart, Allmandring 31, D-70569
Stuttgart, Germany. Phone: 49-711-6855489. Fax: 49-711-6855725. E-mail:
michael.schloemann{at}po.uni-stuttgart.de.
Present address: University Children's Hospital, D-79106
Freiburg, Germany.
Present address: Institute for Crystallography, Free University of
Berlin, D-14195 Berlin, Germany.
Applied and Environmental Microbiology, September 1998, p. 3290-3299, Vol. 64, No. 9
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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