TY - JOUR
T1 - Chitoporin from Serratia marcescens
T2 - Recombinant expression, purification and crystallization
AU - Amornloetwattana, Rawiporn
AU - Robinson, Robert C.
AU - Soysa, Hannadige Sasimali Madusanka
AU - Van Den Berg, Bert
AU - Suginta, Wipa
N1 - Funding Information:
The authors declare no conflicts of interest. We thank the experimental facility and the technical services provided by the Synchrotron Radiation Protein Crystallography Facility of the National Core Facility Program for Biotechnology, Ministry of Science and Technology and the National Synchrotron Radiation Research Centre, a national user facility supported by the Ministry of Science and Technology, Taiwan, and Diamond Light Source, UK. We would like to thank Dr Yoshihito Kitaoku for assisting with data collection and processing, and Professor Takeshi Watanabe, Department of Applied Biological Chemistry, Niigata University, Japan for introducing the chiP gene from the S. marcescens system for functional characterization. We would like to thank Dr David Apps of the University of Edinburgh for critical reading and English improvement of the manuscript.
Funding Information:
The authors declare no conflicts of interest. We thank the experimental facility and the technical services provided by the Synchrotron Radiation Protein Crystallography Facility of the National Core Facility Program for Biotechnology, Ministry of Science and Technology and the National Synchrotron Radiation Research Centre, a national user facility supported by the Ministry of Science and Technology, Taiwan, and Diamond Light Source, UK. We would like to thank Dr Yoshihito Kitaoku for assisting with data collection and processing, and Professor Takeshi Watanabe, Department of Applied Biological Chemistry, Niigata University, Japan for introducing the chiP gene from the S. marcescens system for functional characterization. We would like to thank Dr David Apps of the University of Edinburgh for critical reading and English improvement of the manuscript. WS received funding from Vidyasirimedhi Institute of Science and Technology (VISTEC) and the Thailand Research Fund (TRF) through a Basic Research Grant (Grant No. BRG610008). RA received a full-time MSc scholarship from VISTEC.
Publisher Copyright:
© 2020 International Union of Crystallography. All rights reserved.
PY - 2020/11/1
Y1 - 2020/11/1
N2 - 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 P4 2 2 1 2 and P4 1 2 1 2, 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.
AB - 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 P4 2 2 1 2 and P4 1 2 1 2, 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.
KW - Serratia marcescens
KW - chitin
KW - chitoligosaccharides
KW - chitoporin
KW - outer membrane proteins
KW - sugar transport
KW - sugar-specific porins
UR - http://www.scopus.com/inward/record.url?scp=85095396807&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85095396807&partnerID=8YFLogxK
U2 - 10.1107/S2053230X20013874
DO - 10.1107/S2053230X20013874
M3 - Article
C2 - 33135672
AN - SCOPUS:85095396807
VL - 76
SP - 536
EP - 543
JO - Acta Crystallographica Section F:Structural Biology Communications
JF - Acta Crystallographica Section F:Structural Biology Communications
SN - 1744-3091
ER -