Structure and Mechanism of Pseudomonas aeruginosa PA0254/HudA, a prFMN-Dependent Pyrrole-2-carboxylic Acid Decarboxylase Linked to Virulence

Abstract

The UbiD family of reversible (de)carboxylases depends on the recently discovered prenylated-FMN (prFMN) cofactor for activity. The model enzyme ferulic acid decarboxylase (Fdc1) decarboxylates unsaturated aliphatic acids via a reversible 1,3-cycloaddition process. Protein engineering has extended the Fdc1 substrate range to include (hetero)aromatic acids, although catalytic rates remain poor. This raises the question how efficient decarboxylation of (hetero)aromatic acids is achieved by other UbiD family members. Here, we show that the Pseudomonas aeruginosa virulence attenuation factor PA0254 / HudA is a pyrrole-2-carboxylic acid decarboxylase. The crystal structure of the enzyme in the presence of the reversible inhibitor imidazole reveals a covalent prFMN – imidazole adduct is formed. Substrate screening reveals HudA and selected active site variants can accept a modest range of heteroaromatic compounds, including thiophene-2-carboxylic acid. Together with computational studies, our data suggests prFMN covalent catalysis occurs via electrophilic aromatic substitution and links HudA activity with the inhibitory effects of pyrrole-2-carboxylic acid on P. aeruginosa quorum sensing.

UbiD domain dynamics underpins aromatic decarboxylation

“…This helix has been previously postulated as the point of flexibility in E. coli UbiD 20 and AroY 18 on the basis of the small hinge motions observed by comparing various open state monomers. In the case of PA0254, overlays of the respective apo - and holo -structures also requires a similar hinge motion 19 . In VdcCD, the hinge rotation angle is approximately 32°, with only a negligible translation of the domains (<1 Å).…”

“…A comparison of the Sh VdcC monomers across the Sh VdcCD crystal structures obtained reveals that the prFMN binding domain adopts different conformations with respect to the oligomerisation domain, confirming the existence of distinct open and closed states (Supplementary movie 1 illustrates the motion between the open and closed hexamers). The distinct states occur independent of both prFMN cofactor and VdcD binding (both states occur for the apo- VdcCD complex), in contrast to the apo -open and holo -closed PA0254 structures where domain motion appears linked to cofactor binding 19 . Sh VdcCD structures thus allow for detailed inspection of the transition between the two states.…”

“…1b ) 18 and heteroaromatic acids (Fig. 1c ) 6 , 19 . These all propose formation of a series of covalent adducts between the prFMN C1’ and the substrate Cα ( i.e .…”

“…b A substrate nucleophilic attack mechanism proposed for phenolic substrates 18 . c Electrophilic aromatic substitution mechanism postulated for PA0254-mediated decarboxylation of pyrrole-2-carboxylate 19 . All mechanisms share a common Int2 intermediate with an sp 2 hybridized substrate Cα.…”

“…1d ). However, only the PA0254 (also known as HudA) enzyme has previously been shown to adopt both conformations with the transition between both conformations apparently linked to cofactor binding 19 . Hence, only the dimeric and oxygen-tolerant Fdc1 and PA0254 are representatives of the proposed closed state and are accompanied by substrate or ligand complex structures.…”