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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.

Substrate Specificity of and Product Formation by Muconate Cycloisomerases: an Analysis of Wild-Type Enzymes and Engineered Variants

Martin Dominik Vollmer,1,dagger Helga Hoier,2,Dagger Hans-Jürgen Hecht,3 Ursula Schell,1 Janosch Gröning,1 Adrian Goldman,4,5 and Michael Schlömann1,*

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, FIN-20521 Turku,4 and Department of Biochemistry and Food Chemistry, University of Turku, FIN-20010 Turku,5 Finland

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.

* 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.

dagger Present address: University Children's Hospital, D-79106 Freiburg, Germany.

Dagger 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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