Crystal Structure of a Hidden Protein, YcaC, a Putative Cysteine Hydrolase from Pseudomonas aeruginosa, with and without an Acrylamide Adduct

Abstract

As part of the ongoing effort to functionally and structurally characterize virulence factors in the opportunistic pathogen Pseudomonas aeruginosa, we determined the crystal structure of YcaC co-purified with the target protein at resolutions of 2.34 and 2.56 Å without a priori knowledge of the protein identity or experimental phases. The three-dimensional structure of YcaC adopts a well-known cysteine hydrolase fold with the putative active site residues conserved. The active site cysteine is covalently bound to propionamide in one crystal form, whereas the second form contains an S-mercaptocysteine. The precise biological function of YcaC is unknown; however, related prokaryotic proteins have functions in antibacterial resistance, siderophore production and NADH biosynthesis. Here, we show that YcaC is exceptionally well conserved across both bacterial and fungal species despite being non-ubiquitous. This suggests that whilst YcaC may not be part of an integral pathway, the function could confer a significant evolutionary advantage to microbial life.

Microbial biodegradation of biuret: defining biuret hydrolases within the isochorismatase superfamily

“…Gel filtration analysis suggested that the R. leguminosarum and Herbaspirillum native enzymes were composed of 3 or 4 monomer subunits, and that the Rhodococcus native enzyme was composed of 4 or 5 subunits (Table ). These results are consistent with the tetrameric structure of the R. leguminosarum BH (Esquirol et al ., ) and the multimeric states of other IHL family proteins (Caruthers et al ., ; Goral et al ., ; Grøftehauge et al ., ).…”

The 1.1 Å resolution structure of a periplasmic phosphate-binding protein fromStenotrophomonas maltophilia: a crystallization contaminant identified by molecular replacement using the entire Protein Data Bank

“…In the case of the membrane protein AcrB, the problem is compounded by its ability to crystallize when present in picogram quantities (Psakis et al, 2009). However, in favourable cases these crystallization 'accidents' have led to new structures being solved, such as yeast nicotinamidase (Hu et al, 2005(Hu et al, , 2006(Hu et al, , 2007 and YcaC from both E. coli (Colovos et al, 1998) and P. aeruginosa (Grøftehauge et al, 2015). Crystallization contaminants are often only identified after X-ray data collection and processing, at which point molecular replacement with structures that are expected to have sequence similarity to the target protein proves unsuccessful.…”