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The lactonization of 3-carboxy-cis,cis-mucconate to 4-carboxymuconolactone is catalyzed by β-carboxy-cis,cis-mucconate lactonizing enzyme (CMLE) and is an intermediate step in the catabolism of a variety of aromatic compounds through the β-ketoadipate pathway.
CMLE from Acinetobacter calcoaceticus (Ac-CMLE) was cloned and expressed in E.coli to high yields. Large tetragonal (I4(1)22) crystals grew under conditions which contained Peg6000, LiCl, and NaCitrate. The structure was determined utilizing the dispersive differences from a single derivative (SIR) collected on our home source. Ac-CMLE is a tetramer in solution with an approximate molecular weight of 200,000. Each Ac-CMLE monomer (452 AA) is composed mainly of β-helices and can be separated into three domains. The long helices of each central domain pack against each other to form a large multihelix bundle and the core of the protein. Residues from the presumed active site are donated from all three domains, with the caveat that the three domains originate from three different subunits. As predicted by sequence comparisons Ac-CMLE has high structural homology with members of the Class II Fumarase family of proteins which include the class II fumarases, δ-crystallins, and several lyases such as adenylsuccinate-lyase. There is interest in the enzyme mechanism of CMLEs since it is an intramolecular variant of the fumarate reaction. Structural and biochemical analysis of CMLE and CMLE-Substrate complexes may shed light on which residues are critical to activity in this protein family; as this is still an open question. These studies are now being pursued in the lab.
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