Modulation of the function of small GTPases that regulate vesicular trafficking is a strategy employed by several human pathogens. Legionella pneumophila 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 Legionella pneumophila protein DrrA and of AMP-transfer reactions in general.

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