Characterization of the structure and catalytic activity of Legionella pneumophila VipF

Abstract

The pathogenic bacteria Legionella pneumophila is known to cause Legionnaires' Disease, a severe pneumonia that can be fatal to immunocompromised individuals and the elderly. Shohdy et al. identified the L. pneumophila vacuole sorting inhibitory protein VipF as a putative N-acetyltransferase based on sequence homology. We have characterized the basic structural and functional properties of VipF to confirm this original functional assignment. Sequence conservation analysis indicates two putative CoA-binding regions within VipF. Homology modeling and small angle X-ray scattering suggest a monomeric, dual-domain structure joined by a flexible linker. Each domain contains the characteristic beta-splay motif found in many acetyltransferases, suggesting that VipF may contain two active sites. Docking experiments suggest reasonable acetyl-CoA binding locations within each beta-splay motif. Broad substrate screening indicated that VipF is capable of acetylating chloramphenicol and both domains are catalytically active. Given that chloramphenicol is not known to be N-acetylated, this is a surprising finding suggesting that VipF is capable of O-acetyltransferase activity. Proteins 2016; 84:1422-1430. © 2016 Wiley Periodicals, Inc.

An acetyltransferase effector conserved across Legionella species targets the eukaryotic eIF3 complex to modulate protein translation

“…Among tested substrates, we observed that Lha0223 activity was highest against chloramphenicol, in line with the general activity reported for L. pneumophila VipF/Lpg0103 ( Fig. 1B ) ( 22 , 23 ). However, Lha0223 also demonstrated strong activity against poly-lysine substrate suggesting that it can be active against peptide or protein targets.…”

“…A previous report suggested that both GNAT domains of Lpg0103 are active against chloramphenicol substrate. However, a more recent report established that VipF follows the general trend of tandem GNATs with only the C-terminal domain retaining the catalytic activity ( 22 , 23 ). Despite these insights into the biochemical activity of VipF, the significance of its acetyltransferase activity function for Legionella ’s invasion of the host cell has never been elucidated.…”

Structural basis for the acetylation mechanism of the Legionella effector VipF

“…S2). Young and coworkers found that chloramphenicol is a putative substrate of VipF (Young et al, 2016). Indeed, VipF showed significant activity on chloramphenicol (Supplementary Fig.…”

“…In addition, most of these effectors contain unknown protein sequence domains, except for Lpg1356, which contains multiple Sel1 repeats (Shohdy et al, 2005). Interestingly, VipF has dual GNAT-like domains that catalyze O-linked rather than N-linked acetylation of the putative substrate chloramphenicol (Young et al, 2016). Its enzymatic mechanism, however, has not been fully characterized.…”

The Legionella genus core effectors display functional conservation among orthologs by themselves or combined with an accessory protein

“…Lpg3000 is required for intracellular growth in Hartmannella vermiformis , and lpg0086 is required for intracellular growth in several host cells ( Shames et al., 2017 ; Park et al., 2020b ). In addition, most of these effectors contain no known protein sequence domains, except for lpg1356, which harbors multiple Sel1 repeats and VipF, which contains an acetyltransferase domain and might be involved in membrane trafficking ( Shohdy et al., 2005 ; Young et al., 2016 ).…”