Part:BBa_K5246008

CB2/CB2A HfsH Deacetylase

Usage and Biology

HfsH encodes a cytoplasmic deacetylase of 257 amino acids long. Deacetylase belongs to carbohydrate esterase family 4, catalyze the hydrolysis of N-linked acetyl groups from GlcNAc residues. C.Crescentus HfsH mutants were completely devoid of holdfast material Another C. crescentus CB15 mutant, ΔhfsH (YB2198), was used to study the role of deacethylation in adhesion efficiency. Indeed, this mutant is lacking the gene hfsH, encoding a deacetylase that affects both cohesive and adhesive properties of the holdfast.30 C. crescentus ΔhfsH produces smaller holdfasts compared to the wild-type and the ΔhfaB strains. These fully acetylated holdfasts are not anchored properly to the cell envelope and are shed in the medium. [2] The main strain used in this study was Caulobacter crescentus CB15 ΔhfaB (YB4251),29 a mutant strain from C. crescentus CB15 wild-type (YB135). This mutant has a clean deletion of the hfaB gene and therefore does not synthesize HfaB, one of the holdfast anchor proteins. This strain still produces a holdfast, but is unable to anchor it to the cell envelope. As a consequence, the newly synthesized holdfast is shed in the culture medium and on surfaces. [3]

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 10
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 10
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 10
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 10
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 10
  Illegal NgoMIV site found at 68
  Illegal NgoMIV site found at 92
  Illegal NgoMIV site found at 96
  Illegal NgoMIV site found at 169
  Illegal NgoMIV site found at 295
  Illegal NgoMIV site found at 456
  
• 1000
  COMPATIBLE WITH RFC[1000]

Experimental characterization

Bioinformatic analysis

Conserved domain database analysis suggests that HfsH is part of the carbohydrate esterase 4 superfamily and polysaccharide deacetylase family. Proteins of this family may catalyze the N- or O- deacetylation of a substrate. Protein BLAST results show high similarity to peptidoglycan N-acetylglucosamine deacetylase and other polysaccharide deacetylases. Topology analysis with DeepTMHMM and AlphaFold3 structure showed that HfsH is most probably a globular protein located in the cytoplasm. 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).

HfsH is a globular polysaccharide deacetylase that catalyses the deacetylation of N-acetylglucosamine in the holdfast synthesis pathway, previous research supports our conclusions. [1][2][3]

, Fig. 1. Alphafold 3 structure showing

References

1. Wan, Z. et al. (2013a) ‘The adhesive and cohesive properties of a bacterial polysaccharide adhesin are modulated by a deacetylase’, Molecular Microbiology, 88(3), pp. 486–500. doi:10.1111/mmi.12199.
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.
3. Liu, Q. et al. (2022) ‘The screening and expression of polysaccharide deacetylase from caulobacter crescentus and its function analysis’, Biotechnology and Applied Biochemistry, 70(2), pp. 688–696. doi:10.1002/bab.2390.