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MapA is a histidine acid phosphatase (HAP) from Legionella pneumophila that catalyzes the hydroxylation of a phosphoryl group from phosphomonoesters by an active-site histidine. Several structures of HAPs, including MapA, in complex with the inhibitor tartrate have been solved and the substrate binding tunnel identified; however, the substrate recognition mechanism remains unknown. To gain insight into the mechanism of substrate recognition, the crystal structures of apo-MapA and the MapA D281A mutant in complex with 5'-AMP were solved at 2.2 and 2.6 Å resolution, respectively. The structure of the MapA D281A /5'-AMP complex reveals that the 5'-AMP fits fully into the substrate binding tunnel, with the 2'-hydroxyl group of the ribose moiety stabilized by Glu201 and the adenine moiety sandwiched between His205 and Phe237. This is the second structure of a HAP/AMP complex solved with 5'-AMP binding in a unique manner in the active site. The structure presents a new substrate recognition mechanism of HAPs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The activity of proteins delivered into host cells by the Dot/Icm injection apparatus allows Legionella pneumophila to establish a niche called the Legionella-containing vacuole (LCV), which is permissive for intracellular bacterial propagation. Among these proteins, substrate of Icm/Dot transporter (SidC) anchors to the cytoplasmic surface of the LCV and is important for the recruitment of host endoplasmic reticulum (ER) proteins to this organelle. However, the biochemical function underlying this activity is unknown. Here, we determined the structure of the N-terminal domain of SidC, which has no structural homology to any protein. Sequence homology analysis revealed a potential canonical catalytic triad formed by Cys46, His444, and Asp446 on the surface of SidC. Unexpectedly, we found that SidC is an E3 ubiquitin ligase that uses the C-H-D triad to catalyze the formation of high-molecular-weight polyubiquitin chains through multiple ubiquitin lysine residues. A C46A mutation completely abolished the E3 ligase activity and the ability of the protein to recruit host ER proteins as well as polyubiquitin conjugates to the LCV. Thus, SidC represents a unique E3 ubiquitin ligase family important for phagosomal membrane remodeling by L. pneumophila.