Reaction
Mechanism of Adenylyltransferase DrrA from <i>Legionella pneumophila</i> Elucidated by Time-Resolved Fourier
Transform Infrared Spectroscopy
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Abstract
Modulation
of the function of small GTPases that regulate vesicular
trafficking is a strategy employed by several human pathogens. <i>Legionella pneumophila</i> infects lung macrophages and injects
a plethora of different proteins into its host cell. Among these is
DrrA/SidM, which catalyzes stable adenylylation of Rab1b, a regulator
of endoplasmatic reticulum to Golgi trafficking, and thereby alters
the function and interactions of this small GTPase. We employed time-resolved
FTIR-spectroscopy to monitor the DrrA-catalyzed AMP-transfer to Tyr77
of Rab1b. A transient complex between DrrA, adenylylated Rab1b, and
the pyrophosphate byproduct was resolved, allowing us to analyze the
interactions at the active site. Combination of isotopic labeling
and site-directed mutagenesis allowed us to derive the catalytic mechanism
of DrrA from the FTIR difference spectra. DrrA shares crucial residues
in the ATP-binding pocket with similar AMP-transferring enzymes such
as glutamine synthetase adenylyltransferase or kanamycin nucleotidyltransferase,
but provides the complete active site on a single subunit. We determined
that Asp112 of DrrA functions as the catalytic base for deprotonation
of Tyr77 of Rab1b to enable nucleophilic attack on the ATP. The study
provides detailed understanding of the <i>Legionella pneumophila</i> protein DrrA and of AMP-transfer reactions in general