The atomic-resolution structure of a novel bacterial esterase

Area of Science:

• Enzymology
• Structural Biology
• Biochemistry

Background:

• A novel bacterial esterase, esterase 713, was isolated from an Alcaligenes species, capable of cleaving esters on halogenated cyclic compounds.
• The esterase is encoded by a 1062 base pair gene and possesses a leader sequence, suggesting cytosolic export.
• Over-expression of esterase 713 was achieved in Agrobacterium without its native leader sequence.

Purpose of the Study:

• To determine the crystal structure of esterase 713.
• To elucidate the structural basis for its catalytic activity and compare it with other known esterases.
• To understand the role of specific residues and disulfide bonds in enzyme function.

Main Methods:

• X-ray crystallography was employed to determine the structure of esterase 713 to 1.1 Å resolution using multiple isomorphous replacement.
• Refinement of the crystal structure was performed.
• Binding of a substrate analogue was used to identify the oxyanion hole residues.

Main Results:

• The crystal structure revealed esterase 713 as a dimer with an alpha/beta hydrolase fold.
• The catalytic triad was identified as Ser206-His298-Glu230, with Glu230 located on the beta6 strand, differing from typical esterases.
• The oxyanion hole was formed by Cys71 and Gln207, and Cys71 was found to form a disulfide bond with Cys72.

Conclusions:

• Esterase 713 shares structural similarities with other esterases and lipases despite negligible sequence homology.
• Structural comparison with Rhizomucor miehei lipase confirmed the oxyanion hole residues.
• A disulfide bond between Cys71 and Cys72 is proposed to be essential for a functional active site upon export to the periplasmic space.