Chitoporin fromSerratia marcescens: recombinant expression, purification and crystallization

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 SmChiP, from the outer membrane of S. marcescens. Sequence alignment with genetically characterized ChiPs suggests that SmChiP is more closely related to the monomeric EcChiP from Escherichia coli than to the trimeric VhChiP from Vibrio campbellii. A single crystal of SmChiP 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. SmChiP 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 SmChiP crystal forms contain one molecule per asymmetric unit and that they belong to the tetragonal space groups P42212 and P41212, respectively. The SmChiP crystal has unit-cell parameters a = 82.97, b = 82.97, c = 189.53 Å, α = β = γ = 90°, while the crystal of SmChiP 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 SmChiP in the absence and presence of chitohexaose should reveal the molecular basis of chitin utilization by S. marcescens.

The C2 entity of chitosugars is crucial in molecular selectivity of the Vibrio campbellii chitoporin

“…The difference in the interaction of Vh ChiP with chitohexaose and chitosan hexaose suggests that the nature of the C2 substitution is a key factor in the ability of an approaching sugar molecule to enter the pore. The presence of N-acetyl groups permits pore entry and defines the high specificity of Vh ChiP for acetylated chitooligosaccharides, which are produced by Vh ChiP-expressing marine bacteria such as Vibrio campbelli and also by Vibrio cholera ( 67 ), Serratia marcescens ( 68 ), and Escherichia coli ( 69 ), through enzymic cleavage of available host chitin. Information on the amino acid sequence alignments between the genes of VhChiP and the other chitoporin is available in the original reports ( 16 , 67 , 68 , 69 ).…”

“…The presence of N-acetyl groups permits pore entry and defines the high specificity of Vh ChiP for acetylated chitooligosaccharides, which are produced by Vh ChiP-expressing marine bacteria such as Vibrio campbelli and also by Vibrio cholera ( 67 ), Serratia marcescens ( 68 ), and Escherichia coli ( 69 ), through enzymic cleavage of available host chitin. Information on the amino acid sequence alignments between the genes of VhChiP and the other chitoporin is available in the original reports ( 16 , 67 , 68 , 69 ).…”

Development of a microbial protease for composting swine carcasses, optimization of its production and elucidation of its catalytic hydrolysis mechanism

“…Therefore, strain DB1 was defined as Serratia marcescens sp. It is noteworthy that Serratia marcescens has been widely reported in chitin production, detergent production, bioremediation and hydrolase production [23][24][25]32]. However, there are few reports about protease-producing Serratia marcescens isolated from composting.…”