The structure of Pseudomonas P51 Cl-muconate lactonizing enzyme: co-evolution of structure and dynamics with the dehalogenation function.
Bacterial muconate lactonizing enzymes (MLEs) catalyze the conversion of cis,cis-muconate as a part of the beta-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 A 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.
Kajander
Tommi
T
Research Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, FIN-00014 Helsinki, Finland.
Lehtiö
Lari
L
Schlömann
Michael
M
Goldman
Adrian
A
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D006219:Halogens / Q000737:chemistry; D006867:Hydrolases / Q000378:metabolism; D019753:Intramolecular Lyases / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D006219:Halogens; D006867:Hydrolases; D019753:Intramolecular Lyases; C021811:muconate cycloisomerase