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

Open Access
Publisher Website
Google Scholar
Abstract
Bacterial muconate lactonizing enzymes (MLEs) catalyze the conversion of cis,cis‐muconate as a part of the β‐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.