Difference between revisions of "Part:BBa K5246001"
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===Experimental characterization===
 
===Experimental characterization===
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====Cloning part====
  
 
===References===
 
===References===

Revision as of 18:25, 28 September 2024


CB2/CB2A HfsA Part of export protein complex

Introduction

Usage and Biology

Gene HfsA from Caulobacter crescentus encodes a protein of 502 amino acids from complex that in combination with HfsB is responsible for controlled polymerisation of holdfast pollysaccharide. HfsA is a polysaccharide copolymerase Wzz

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 388
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 64
    Illegal NgoMIV site found at 256
    Illegal NgoMIV site found at 265
    Illegal NgoMIV site found at 837
    Illegal NgoMIV site found at 1000
  • 1000
    COMPATIBLE WITH RFC[1000]

Bioinformatical characterization

Bioinformatic analysis

Conservative Domain Database analysis revealed that the HfsA protein contains domains characteristic of the GumC superfamily, which typically function in exopolysaccharide export within the cell wall or membrane. Additionally, it shows significant similarity to the PEP-CTERM superfamily, proteins that are generally involved in determining polysaccharide chain length.

NCBI protein BLAST analysis revealed significant similarities between HfsA and a capsular polysaccharide biosynthesis protein from the ABC transporter family of Caldimonas thermodepolymerans. Using the DeepTMHMM tool to analyze its transmembrane structure, it was predicted that HfsA spans the membrane twice, embedding itself firmly in the cell envelope.

With AlphaFold3, we assessed different configurations of HfsA subunits, focusing on ipTM and pTM scores. 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 We hypothesise that HfsA transmembrane protein consists of 8 subunits with confidence scores of ipTM = 0.72 and pTM = 0.73 (Fig.1).

Based on this data, we hypothesize that HfsA is a transmembrane protein responsible for exporting polysaccharides from the cell. Similar findings were proposed by earlier research. [1][2][3][4]

hfsa-1.png, hfsa-2.png
Fig. 1. Alphafold 3 structure showing assembled hfsA protein of 8 subunits. On the left-folded structure with confidence scores above, on the right-predicted aligned error plot of the structure

Experimental characterization

Cloning part

References

1. Kurtz, H.D. and Smith, J. (1994) ‘The Caulobacter crescentushold fast: Identification of holdfast attachment complex genes’, FEMS Microbiology Letters, 116(2), pp. 175–182. doi:10.1111/j.1574-6968.1994.tb06697.x.
2. Javens, J. et al. (2013) ‘Bypassing the need for subcellular localization of a polysaccharide export‐anchor complex by overexpressing its protein subunits’, Molecular Microbiology, 89(2), pp. 350–371. doi:10.1111/mmi.12281.
3. Smith, C.S. et al. (2003) ‘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.
4. Toh, E., Kurtz, H.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. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2580695/