Chitoporin from Serratia marcescens: recombinant expression, purification and crystallization. Issue 11 (2nd November 2020) - Amornloetwattana, Rawiporn
Abstract : Serratia marcescens is an opportunistic pathogen that can use chitin‐enriched nutrients as an alternative energy source. We identified a chitoporin (ChiP), termed SmChiP, from the outer membrane of S. marcescens and report its crystallization without and with chitohexaose. Initial assessment of the complex structure revealed electron density for the sugar ligand. Abstract : Serratia marcescens is an opportunistic pathogen that commonly causes hospital‐acquired infections and can utilize chitin‐enriched nutrients as an alternative energy source. This study reports the identification of a chitoporin (ChiP), termed Sm ChiP, from the outer membrane of S. marcescens . Sequence alignment with genetically characterized ChiPs suggests that Sm ChiP is more closely related to the monomeric Ec ChiP from Escherichia coli than to the trimeric Vh ChiP from Vibrio campbellii . A single crystal of Sm ChiP grown under the condition 22%( w / v ) PEG 8000, 0.1 M calcium acetate, 0.1 M MES pH 6.0 diffracted X‐ray synchrotron radiation to 1.85 Å resolution. Sm ChiP co‐crystallized with chitohexaose under the condition 19%( w / v ) PEG 1500, 2 M ammonium phosphate monobasic, 0.1 M HEPES pH 7.0 diffracted X‐rays to 2.70 Å resolution. Preliminary crystallographic analysis shows that both Sm ChiP crystal forms contain one molecule per asymmetric unit and that they belong to the tetragonal space groups P 42 21 2 and P 41 21 2, respectively. The Sm ChiP crystal has unit‐cell parameters a = 82.97, b = 82.97, c = 189.53 Å, α = β = γ = 90°, while the crystal of Sm ChiP in complex with chitohexaose has unit‐cell parameters a = 73.24, b = 73.24, c = 213.46 Å, α = β = γ = 90°. Initial assessment of the complex structure clearly revealed electron density for the sugar ligand. Structure determination of Sm ChiP in the absence and presence of chitohexaose should reveal the molecular basis of chitin utilization by S. marcescens .