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Protein Science (2003), 12:1855-1864.
Copyright © 2003 The Protein Society

The structure of Pseudomonas P51 Cl-muconate lactonizing enzyme: Co-evolution of structure and dynamics with the dehalogenation function

Tommi Kajander1,3, Lari Lehtiö1,3, Michael Schlömann2 and Adrian Goldman1

1 Research Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, FIN-00014 Helsinki, Finland
2 Interdisziplinäres Ökologisches Zentrum, TU Bergakademie Freiberg, D-09599 Freiberg, Germany

Reprint requests to: Adrian Goldman, Research Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, P.O. Box 65, FIN-00014 Helsinki, Finland; e-mail: adrian.goldman{at}helsinki.fi; fax: 358-9-191-59940.

Bacterial muconate lactonizing enzymes (MLEs) catalyze the conversion of cis,cis-muconate as a part of the ß-ketoadipate pathway, and some MLEs are also able to dehalogenate chlorinated muconates (Cl-MLEs). The basis for the Cl-MLEs dehalogenating activity is still unclear. To further elucidate the differences between MLEs and Cl-MLEs, we have solved the structure of Pseudomonas P51 Cl-MLE at 1.95 Å resolution. Comparison of Pseudomonas MLE and Cl-MLE structures reveals the presence of a large cavity in the Cl-MLEs. The cavity may be related to conformational changes on substrate binding in Cl-MLEs, at Gly52. Site-directed mutagenesis on Pseudomonas MLE core positions to the equivalent Cl-MLE residues showed that the variant Thr52Gly was rather inactive, whereas the Thr52Gly-Phe103Ser variant had regained part of the activity. These residues form a hydrogen bond in the Cl-MLEs. The Cl-MLE structure, as a result of the Thr-to-Gly change, is more flexible than MLE: As a mobile loop closes over the active site, a conformational change at Gly52 is observed in Cl-MLEs. The loose packing and structural motions in Cl-MLE may be required for the rotation of the lactone ring in the active site necessary for the dehalogenating activity of Cl-MLEs. Furthermore, we also suggest that differences in the active site mobile loop sequence between MLEs and Cl-MLEs result in lower active site polarity in Cl-MLEs, possibly affecting catalysis. These changes could result in slower product release from Cl-MLEs and make it a better enzyme for dehalogenation of substrate.

Keywords: Cl-muconate lactonizing enzyme; conformational changes; mechanism; structure/function studies; crystallography; mutagenesis; dehalogenation


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