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Proteins. 2011 Nov;79(11):3180-92. doi: 10.1002/prot.23150. Epub 2011 Aug 30.

Effects of the Val211Gly substitution on molecular dynamics of the CMY-2 cephalosporinase: implications on hydrolysis of expanded-spectrum cephalosporins.

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Laboratory of Bacteriology, Hellenic Pasteur Institute, 11521 Athens, Greece.


CMY-30, a naturally occurring class C β-lactamase differing from the Citrobacter freundii-derived CMY-2 by a Val211Gly substitution in the Ω-loop, exhibits increased hydrolytic efficiency against ceftazidime and cefotaxime. Kinetic constants of CMY-2 and CMY-30 against the latter substrates suggested that the improved efficiency of the Gly211 variant was due to an increase in k(cat). The structural basis of the increased turn-over rates of oxyimino-cephalosporins caused by Val211Gly was studied using 5 ns molecular dynamics simulations of CMY-2 and CMY-30 in their free forms and in covalent complexes with ceftazidime (acyl-enzyme) as well as a boronic acid analogue of ceftazidime (deacylation transition state). Analysis of thermal factors indicated that Val211Gly increased the flexibility of the Ω-loop/H7-helix and the Q120-loop formed by amino acids 112-125, and also altered the vibrations of the H10-helix/R2-loop. Structural elements containing the catalytic residues remained relatively rigid except Tyr150 in acyl-enzyme species. Regions exhibiting altered flexibility due to the substitution appear to move in a concerted manner in both enzymes. This movement was more intense in CMY-30 and also at directions different to those observed for CMY-2. Additionally, it appeared that the Val211Gly increased the available space for the accommodation of the R1 side chain of ceftazidime. These findings are likely associated with the significantly increased vibrations of the bound compounds observed in CMY-30 complexes. Therefore, the extended spectrum properties of CMY-30 seem to arise through a complex process implicating changes in protein movement and in the mode of substrate accommodation.

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