Difference between revisions of "Part:BBa K5246003"
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===Usage and Biology===
 
===Usage and Biology===
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Gene from Caulobacter crescentud HfsC, encodes a protein of 422aa that polymerases repeats of monomers into a mature holdfast polymer, deletion of polysaccharide polymerase gene hfsC in C.Crescentus didn't cause holdfast synthesis defects, because of its paralogue - HfsI. Double mutants of HfsC and HfsI cause severe holfast synthesis defects
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Gene from Caulobacter crescentus HfsC, encodes a protein of 422 amino acids that polymerases repeats of monomers into a mature holdfast polymer, deletion of polysaccharide polymerase gene hfsC in C.Crescentus didn't cause holdfast synthesis defects, because of its paralogue - HfsI. Double mutants of HfsC and HfsI cause severe holdfast synthesis defects
  
 
===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K5246003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5246003 SequenceAndFeatures</partinfo>
  
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===Functional Parameters===
 
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<partinfo>BBa_K5246003 parameters</partinfo>
 
  
 
===Experimental characterization===
 
===Experimental characterization===
  
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====Bioinformatic analysis====
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CDD analysis showed that only part of the protein has any similarities with established conservative domains. The predicted domain is part of the O-antigen ligase family, which is a group of proteins responsible for outer membrane lipopolysaccharide synthesis in E.Coli.
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 +
Protein BLAST also showed partial similarities with E.Coli O-antigen ligases suggested by the CDD analysis.
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 +
DeepTMHMM predicted that the protein is embedded in the membrane, crossing it approximately 12 times. This prediction is supported by structural evidence from AlphaFold3, which shows 12 helices. A pTM score above 0.5 suggests that the predicted overall structure may closely resemble the true protein fold, while ipTM indicates the accuracy of the subunit positioning within the complex. Values higher than 0.8 represent confident, high-quality predictions (Fig.1).
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 +
Considering the data, HfsC is most probably a membrane protein that functions as a polysaccharide ligase due to its similarity to O-antigen ligase; this hypothesis is further supported by earlier research. [1][2]
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<center> https://static.igem.wiki/teams/5246/registry/hfsc.png </center>
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<center> <b> Fig. 1. </b> Alphafold 3 structure </center>
  
 
===References===
 
===References===
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1. Smith, C.S. et al. (2003a) ‘Identification of genes required for synthesis of the adhesive holdfast in            Caulobacter crescentus’, Journal of Bacteriology, 185(4), pp. 1432–1442. doi:10.1128/jb.185.4.1432-1442.2003.
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<br>
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2. Toh, E., Kurtz, Harry D. and Brun, Y.V. (2008) ‘Characterization of the Caulobacter crescentus holdfast polysaccharide biosynthesis pathway reveals significant redundancy in the initiating glycosyltransferase and polymerase steps’, Journal of Bacteriology, 190(21), pp. 7219–7231. doi:10.1128/jb.01003-08.

Revision as of 13:21, 27 September 2024


CB2/CB2A HfsC Polysaccharide polymerase

Introduction

Usage and Biology

Gene from Caulobacter crescentus HfsC, encodes a protein of 422 amino acids that polymerases repeats of monomers into a mature holdfast polymer, deletion of polysaccharide polymerase gene hfsC in C.Crescentus didn't cause holdfast synthesis defects, because of its paralogue - HfsI. Double mutants of HfsC and HfsI cause severe holdfast synthesis defects

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1072
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1072
    Illegal NotI site found at 745
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 491
    Illegal BglII site found at 863
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1072
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1072
    Illegal NgoMIV site found at 12
    Illegal NgoMIV site found at 109
    Illegal NgoMIV site found at 732
  • 1000
    COMPATIBLE WITH RFC[1000]


Experimental characterization

Bioinformatic analysis

CDD analysis showed that only part of the protein has any similarities with established conservative domains. The predicted domain is part of the O-antigen ligase family, which is a group of proteins responsible for outer membrane lipopolysaccharide synthesis in E.Coli.

Protein BLAST also showed partial similarities with E.Coli O-antigen ligases suggested by the CDD analysis.

DeepTMHMM predicted that the protein is embedded in the membrane, crossing it approximately 12 times. This prediction is supported by structural evidence from AlphaFold3, which shows 12 helices. A pTM score above 0.5 suggests that the predicted overall structure may closely resemble the true protein fold, while ipTM indicates the accuracy of the subunit positioning within the complex. Values higher than 0.8 represent confident, high-quality predictions (Fig.1).

Considering the data, HfsC is most probably a membrane protein that functions as a polysaccharide ligase due to its similarity to O-antigen ligase; this hypothesis is further supported by earlier research. [1][2]

hfsc.png
Fig. 1. Alphafold 3 structure

References

1. Smith, C.S. et al. (2003a) ‘Identification of genes required for synthesis of the adhesive holdfast in Caulobacter crescentus’, Journal of Bacteriology, 185(4), pp. 1432–1442. doi:10.1128/jb.185.4.1432-1442.2003.
2. Toh, E., Kurtz, Harry D. and Brun, Y.V. (2008) ‘Characterization of the Caulobacter crescentus holdfast polysaccharide biosynthesis pathway reveals significant redundancy in the initiating glycosyltransferase and polymerase steps’, Journal of Bacteriology, 190(21), pp. 7219–7231. doi:10.1128/jb.01003-08.