Mechanism and Diversity
of the Erythromycin Esterase Family of Enzymes
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Abstract
Macrolide antibiotics such as azithromycin and erythromycin
are mainstays of modern antibacterial chemotherapy, and like all antibiotics,
they are vulnerable to resistance. One mechanism of macrolide resistance
is via drug inactivation: enzymatic hydrolysis of the macrolactone
ring catalyzed by erythromycin esterases, EreA and EreB. A genomic
enzymology approach was taken to gain insight into the catalytic mechanisms
and origins of Ere enzymes. Our analysis reveals that erythromycin
esterases comprise a separate group in the hydrolase superfamily,
which includes homologues of uncharacterized function found on the
chromosome of <i>Bacillus cereus</i>, Bcr135 and Bcr136,
whose three-dimensional structures have been determined. Biochemical
characterization of Bcr136 confirms that it is an esterase that is,
however, unable to inactivate macrolides. Using steady-state kinetics,
homology-based structure modeling, site-directed mutagenesis, solvent
isotope effect studies, pH, and inhibitor profiling performed in various
combinations for EreA, EreB, and Bcr136 enzymes, we identified the
active site and gained insight into some catalytic features of this
novel enzyme superfamily. We rule out the possibility of a Ser/Thr
nucleophile and show that one histidine, H46 (EreB numbering), is
essential for catalytic function. This residue is proposed to serve
as a general base in activation of a water molecule as the reaction
nucleophile. Furthermore, we show that EreA, EreB, and Bcr136 are
distinct, with only EreA inhibited by chelating agents and hypothesized
to contain a noncatalytic metal. Detailed characterization of these
esterases allows for a direct comparison of the resistance determinants,
EreA and EreB, with their prototype, Bcr136, and for the discussion
of their potential connections