Chitoporin fromSerratia marcescens: recombinant expression, purification and crystallization

Abstract

Serratia marcescensis 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), termedSmChiP, from the outer membrane ofS. marcescens. Sequence alignment with genetically characterized ChiPs suggests thatSmChiP is more closely related to the monomericEcChiP fromEscherichia colithan to the trimericVhChiP fromVibrio campbellii. A single crystal ofSmChiP grown under the condition 22%(w/v) PEG 8000, 0.1 Mcalcium acetate, 0.1 MMES pH 6.0 diffracted X-ray synchrotron radiation to 1.85 Å resolution.SmChiP co-crystallized with chitohexaose under the condition 19%(w/v) PEG 1500, 2 Mammonium phosphate monobasic, 0.1 MHEPES pH 7.0 diffracted X-rays to 2.70 Å resolution. Preliminary crystallographic analysis shows that bothSmChiP crystal forms contain one molecule per asymmetric unit and that they belong to the tetragonal space groupsP42212 andP41212, respectively. TheSmChiP crystal has unit-cell parametersa= 82.97,b= 82.97,c= 189.53 Å, α = β = γ = 90°, while the crystal ofSmChiP in complex with chitohexaose has unit-cell parametersa = 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 ofSmChiP in the absence and presence of chitohexaose should reveal the molecular basis of chitin utilization byS. marcescens.