Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site
van der Oost, J
Nature Publishing Group
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A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain pnitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold 2 enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions.
This work was supported by the Hotzyme project (grant agreement no. 265933) financed by the European Union 7th Framework Programme FP7/2007-2013. WF is funded by a BBSRC PhD studentship. MI would like to thank the BBSRC funded ERA-IB grant BB/L002035/1 and the University of Exeter for support. The authors would like to thank the Diamond Synchrotron Light Source for access to beamline I03 (proposals No. MX8889 and No. MX11945) and the beamline scientists for assistance. The work of ML was funded by the Graduate School VLAG Wageningen, the Netherland
Vol. 6, Article number: 25542