Difference between revisions of "Part:BBa K5246017"
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===Usage and Biology===
 
===Usage and Biology===
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The <i>HfsE</i> gene encodes a 518 amino acid protein in <i>Hirschia baltica</i>, identified as a putative integral membrane glycotransferase. The primary role of this protein is to transfer a glucose-1-phosphate from uridine diphosphate glucose (UDP-glucose) or to an undecaprenyl phosphate (UP), which is a crucial lipid carrier that facilitates the transfer of cell wall intermediates across the cytoplasmic membrane in bacteria. This reaction represents the first step in the synthesis of the holdfast polysaccharide repeat units in the cytoplasm. [https://parts.igem.org/Part:BBa_K471901].
+
The <i>HfsE</i> gene encodes a 518 amino acid protein in <i>Hirschia baltica</i>, identified as a putative integral membrane glycotransferase. The primary role of this protein is to transfer a glucose-1-phosphate from uridine diphosphate glucose (UDP-glucose) or to an undecaprenyl phosphate (UP), which is a crucial lipid carrier that facilitates the transfer of cell wall intermediates across the cytoplasmic membrane in bacteria. This reaction represents the first step in synthesizing the holdfast polysaccharide repeat units in the cytoplasm. [https://parts.igem.org/Part:BBa_K471901].
  
  
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===Functional Parameters===
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===Experimental characterization===
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<partinfo>BBa_K5246017 parameters</partinfo>
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<h3>Experimental characterization</h3>
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====Bioinformatic analysis====
  
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According to CDD, protein is similar to undecaprenyl-phosphate glucose phosphotransferases found in E. coli. Most of the genes for these proteins are found within large operons dedicated to the production of complex exopolysaccharides, such as the enterobacterial O-antigen. It also has some overlap with other bacterial sugar-transferases.
 +
 +
Protein BLAST further supports the prediction of HfsE being a UDP-glucose-carrier transferase because of its similarities with multiple glucose transferases.
 +
 +
HfsE is likely a transmembrane protein that traverses the membrane six times, with a portion of it exposed on the cytoplasmic side.
 +
 +
AlphaFold 3 structure confidence scores suggest that it is a protein made mostly of alpha 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 prediction.
 +
 +
Altogether, HfsE is a membrane protein that is responsible for the first glucose addition to an undecaprenyl-phosphate lipid carrier in the holdfast synthesis pathway, similar to bacterial glucose transferases. Analogous HfsE function is proposed in the literature by earlier research. [1][2][3]
  
 
===References===
 
===References===
−
1. Chepkwony, N.K., Hardy, G.G. and Brun, Y.V. (2022a) ‘HFAE is a component of the holdfast anchor complex that tethers the holdfast adhesin to the cell envelope’, Journal of Bacteriology, 204(11). doi:10.1128/jb.00273-22.
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1. Chepkwony, N.K., Hardy, G.G. and Brun, Y.V. (2022) ‘HFAE is a component of the holdfast anchor complex that tethers the holdfast adhesin to the cell envelope’, Journal of Bacteriology, 204(11). doi:10.1128/jb.00273-22.
 +
<br>
 +
2. 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, 190(21), pp. 7219–7231. doi:10.1128/jb.01003-08.
 +
<br>
 +
3. Patel, K.B. et al. (2012) ‘Functional characterization of UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferases of Escherichia coli and Caulobacter crescentus’, Journal of Bacteriology, 194(10), pp. 2646–2657. doi:10.1128/jb.06052-11.
 +
<br>
 +
4. Chepkwony, N.K., Berne, C. and Brun, Y.V. (2019) ‘Comparative analysis of ionic strength tolerance between freshwater and marine Caulobacterales adhesins’, Journal of Bacteriology, 201(18). doi:10.1128/jb.00061-19.  
 
<br>
 
<br>
−
2. Toh, E., Kurtz, H.D. and Brun, Y.V. (2008a) ‘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.
+
5. Hershey, D.M., Fiebig, A. and Crosson, S. (2019) ‘A genome-wide analysis of adhesion in Caulobacter crescentus identifies new regulatory and biosynthetic components for holdfast assembly’, mBio, 10(1). doi:10.1128/mbio.02273-18.

Revision as of 10:58, 28 September 2024


HB HfsE glycosyltransferase

Introduction

Usage and Biology

The HfsE gene encodes a 518 amino acid protein in Hirschia baltica, identified as a putative integral membrane glycotransferase. The primary role of this protein is to transfer a glucose-1-phosphate from uridine diphosphate glucose (UDP-glucose) or to an undecaprenyl phosphate (UP), which is a crucial lipid carrier that facilitates the transfer of cell wall intermediates across the cytoplasmic membrane in bacteria. This reaction represents the first step in synthesizing the holdfast polysaccharide repeat units in the cytoplasm. [1].


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 48
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 214
    Illegal AgeI site found at 1381
  • 1000
    COMPATIBLE WITH RFC[1000]


Experimental characterization

Bioinformatic analysis

According to CDD, protein is similar to undecaprenyl-phosphate glucose phosphotransferases found in E. coli. Most of the genes for these proteins are found within large operons dedicated to the production of complex exopolysaccharides, such as the enterobacterial O-antigen. It also has some overlap with other bacterial sugar-transferases.

Protein BLAST further supports the prediction of HfsE being a UDP-glucose-carrier transferase because of its similarities with multiple glucose transferases.

HfsE is likely a transmembrane protein that traverses the membrane six times, with a portion of it exposed on the cytoplasmic side.

AlphaFold 3 structure confidence scores suggest that it is a protein made mostly of alpha 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 prediction.

Altogether, HfsE is a membrane protein that is responsible for the first glucose addition to an undecaprenyl-phosphate lipid carrier in the holdfast synthesis pathway, similar to bacterial glucose transferases. Analogous HfsE function is proposed in the literature by earlier research. [1][2][3]

References

1. Chepkwony, N.K., Hardy, G.G. and Brun, Y.V. (2022) ‘HFAE is a component of the holdfast anchor complex that tethers the holdfast adhesin to the cell envelope’, Journal of Bacteriology, 204(11). doi:10.1128/jb.00273-22.
2. 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, 190(21), pp. 7219–7231. doi:10.1128/jb.01003-08.
3. Patel, K.B. et al. (2012) ‘Functional characterization of UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferases of Escherichia coli and Caulobacter crescentus’, Journal of Bacteriology, 194(10), pp. 2646–2657. doi:10.1128/jb.06052-11.
4. Chepkwony, N.K., Berne, C. and Brun, Y.V. (2019) ‘Comparative analysis of ionic strength tolerance between freshwater and marine Caulobacterales adhesins’, Journal of Bacteriology, 201(18). doi:10.1128/jb.00061-19.
5. Hershey, D.M., Fiebig, A. and Crosson, S. (2019) ‘A genome-wide analysis of adhesion in Caulobacter crescentus identifies new regulatory and biosynthetic components for holdfast assembly’, mBio, 10(1). doi:10.1128/mbio.02273-18.