Difference between revisions of "Part:BBa K5246006"

Revision as of 13:39, 27 September 2024

CB2/CB2A HfsF Flippase

Introduction

Usage and Biology

Encodes an integral membrane protein of 480 amino acids with 14 predicted transmembrane helices. Possesses similarities to Wzx flippases (formerly known as RfbX) that catalyze the translocation of undecaprenol diphosphate-linked K-repeating units formed at the cytoplasmic side of the inner membrane across this membrane.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 1033
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 1033
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 93
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 1033
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 1033
Illegal NgoMIV site found at 343
Illegal NgoMIV site found at 451
Illegal NgoMIV site found at 733
Illegal NgoMIV site found at 790
Illegal NgoMIV site found at 834
Illegal NgoMIV site found at 865
Illegal NgoMIV site found at 1089
Illegal AgeI site found at 151
1000
COMPATIBLE WITH RFC[1000]

Experimental characterization

Bioinformatic analysis

HfsF is a flippase with domains, partly similar to some polysaccharide synthesis pathway proteins in bacteria, as it was shown by CDD analysis. Protein BLAST indicates high similarities to MOP superfamily flippases involved in peptidoglycan synthesis. DeepTMHMM predictions suggest that the protein is located in the membrane, spanning it multiple times. Considering high AlphaFold 3 structure confidence scores, it seems probable that hfsF is made 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 predictions (Fig.1).

All in all, HfsF is a flippase located in the membrane that is responsible for oligosaccharide transfer from the cytoplasmic to the periplasmic side of the inner membrane. This flippase is similar to flippases involved in peptidoglycan synthesis. Our results align with prior studies. [1][2][3]

Fig. 1. AlphaFold 3 structure showing

References

1. 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.
2. Hardy, G.G. et al. (2018) ‘Mutations in sugar-nucleotide synthesis genes restore holdfast polysaccharide anchoring to Caulobacter crescentus holdfast anchor mutants’, Journal of Bacteriology, 200(3). doi:10.1128/jb.00597-17.
3. 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.

@@ Line 7: / Line 7: @@
 ===Usage and Biology===
-Encodes an integral membrane protein of 480 aa with 14 predicted transmembrane helices. Posseses similarities to  Wzx flippases (formerly known as RfbX) that catalyze the translocation of undecaprenol diphosphate-linked K-repeating units formed at the cytoplasmic side of the inner membrane across this membrane
+Encodes an integral membrane protein of 480 amino acids with 14 predicted transmembrane helices. Possesses similarities to  Wzx flippases (formerly known as RfbX) that catalyze the translocation of undecaprenol diphosphate-linked K-repeating units formed at the cytoplasmic side of the inner membrane across this membrane.
 ===Sequence and Features===
 <partinfo>BBa_K5246006 SequenceAndFeatures</partinfo>
-===Functional Parameters===
-<partinfo>BBa_K5246006 parameters</partinfo>
 ===Experimental characterization===
+====Bioinformatic analysis====
+HfsF is a flippase with domains, partly similar to some polysaccharide synthesis pathway proteins in bacteria, as it was shown by CDD analysis.
+Protein BLAST indicates high similarities to MOP superfamily flippases involved in peptidoglycan synthesis.
+DeepTMHMM predictions suggest that the protein is located in the membrane, spanning it multiple times.
+Considering high AlphaFold 3 structure confidence scores, it seems probable that hfsF is made 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 predictions (Fig.1).
+All in all, HfsF is a flippase located in the membrane that is responsible for oligosaccharide transfer from the cytoplasmic to the periplasmic side of the inner membrane. This flippase is similar to flippases involved in peptidoglycan synthesis. Our results align with prior studies. [1][2][3]
+<center> https://static.igem.wiki/teams/5246/registry/hfsf.png </center>
+<center> <b> Fig. 1. </b> AlphaFold 3 structure showing  </center>
 ===References===
+. 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.
+<br>
+. Hardy, G.G. et al. (2018) ‘Mutations in sugar-nucleotide synthesis genes restore holdfast polysaccharide anchoring to Caulobacter crescentus holdfast anchor mutants’, Journal of Bacteriology, 200(3). doi:10.1128/jb.00597-17.
+<br>
+. 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.