Identification of essential amino acids in the bacterial α-mannosyltransferase AceA
The α-mannosyltransferase AceA from Acetobacter xylinum belongs to the CaZY family 4 of retaining glycosyltransferases. We have identified a series of either highly conserved or invariant residues that are found in all family 4 enzymes as well as other retaining glycosyltransferases. These residues...
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2000
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paper:paper_00219258_v275_n51_p40568_Abdian2023-06-08T14:43:19Z Identification of essential amino acids in the bacterial α-mannosyltransferase AceA Abdian, Patricia Ielpi, Luis Geremía, Roberto Alejandro alpha mannosyltransferase acea bacterial enzyme essential amino acid glucosyltransferase mannosyltransferase unclassified drug Acetobacter amino acid substitution article cellular distribution enzyme activity enzyme analysis enzyme structure nonhuman priority journal structure analysis Acetobacter Amino Acids, Essential Catalysis Mannosyltransferases Nuclear Magnetic Resonance, Biomolecular Protein Structure, Secondary Recombinant Proteins Acetobacter Bacteria (microorganisms) Escherichia coli Gluconacetobacter xylinus Xanthomonas campestris The α-mannosyltransferase AceA from Acetobacter xylinum belongs to the CaZY family 4 of retaining glycosyltransferases. We have identified a series of either highly conserved or invariant residues that are found in all family 4 enzymes as well as other retaining glycosyltransferases. These residues included Glu-287 and Glu-295, which comprise an EX7E motif and have been proposed to be involved in catalysis. Alanine replacements of each conserved residue were constructed by site-directed mutagenesis. The mannosyltransferase activity of each mutant was examined by both an in vitro transferase assay using recombinant mutant AceA expressed in Escherichia coli and by an in vivo rescue assay by expressing the mutant AceA in a Xanthomonas campestris gumH- strain. We found that only mutants K211A and E287A lost all detectable activity both in vitro and in vivo, whereas E295A retained residual activity in the more sensitive in vivo assay. H127A and S162A each retained reduced but significant activities both in vitro and in vivo. Secondary structure predictions of AceA and subsequent comparison with the crystal structures of the T4 β-glucosyltransferase and MurG suggest that AceA Lys-211 and Glu-295 are involved in nucleotide sugar donor binding, leaving Glu-287 of the EX7E as a potential catalytic residue. Fil:Abdian, P.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Ielpi, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Geremia, R.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 2000 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00219258_v275_n51_p40568_Abdian http://hdl.handle.net/20.500.12110/paper_00219258_v275_n51_p40568_Abdian |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
alpha mannosyltransferase acea bacterial enzyme essential amino acid glucosyltransferase mannosyltransferase unclassified drug Acetobacter amino acid substitution article cellular distribution enzyme activity enzyme analysis enzyme structure nonhuman priority journal structure analysis Acetobacter Amino Acids, Essential Catalysis Mannosyltransferases Nuclear Magnetic Resonance, Biomolecular Protein Structure, Secondary Recombinant Proteins Acetobacter Bacteria (microorganisms) Escherichia coli Gluconacetobacter xylinus Xanthomonas campestris |
| spellingShingle |
alpha mannosyltransferase acea bacterial enzyme essential amino acid glucosyltransferase mannosyltransferase unclassified drug Acetobacter amino acid substitution article cellular distribution enzyme activity enzyme analysis enzyme structure nonhuman priority journal structure analysis Acetobacter Amino Acids, Essential Catalysis Mannosyltransferases Nuclear Magnetic Resonance, Biomolecular Protein Structure, Secondary Recombinant Proteins Acetobacter Bacteria (microorganisms) Escherichia coli Gluconacetobacter xylinus Xanthomonas campestris Abdian, Patricia Ielpi, Luis Geremía, Roberto Alejandro Identification of essential amino acids in the bacterial α-mannosyltransferase AceA |
| topic_facet |
alpha mannosyltransferase acea bacterial enzyme essential amino acid glucosyltransferase mannosyltransferase unclassified drug Acetobacter amino acid substitution article cellular distribution enzyme activity enzyme analysis enzyme structure nonhuman priority journal structure analysis Acetobacter Amino Acids, Essential Catalysis Mannosyltransferases Nuclear Magnetic Resonance, Biomolecular Protein Structure, Secondary Recombinant Proteins Acetobacter Bacteria (microorganisms) Escherichia coli Gluconacetobacter xylinus Xanthomonas campestris |
| description |
The α-mannosyltransferase AceA from Acetobacter xylinum belongs to the CaZY family 4 of retaining glycosyltransferases. We have identified a series of either highly conserved or invariant residues that are found in all family 4 enzymes as well as other retaining glycosyltransferases. These residues included Glu-287 and Glu-295, which comprise an EX7E motif and have been proposed to be involved in catalysis. Alanine replacements of each conserved residue were constructed by site-directed mutagenesis. The mannosyltransferase activity of each mutant was examined by both an in vitro transferase assay using recombinant mutant AceA expressed in Escherichia coli and by an in vivo rescue assay by expressing the mutant AceA in a Xanthomonas campestris gumH- strain. We found that only mutants K211A and E287A lost all detectable activity both in vitro and in vivo, whereas E295A retained residual activity in the more sensitive in vivo assay. H127A and S162A each retained reduced but significant activities both in vitro and in vivo. Secondary structure predictions of AceA and subsequent comparison with the crystal structures of the T4 β-glucosyltransferase and MurG suggest that AceA Lys-211 and Glu-295 are involved in nucleotide sugar donor binding, leaving Glu-287 of the EX7E as a potential catalytic residue. |
| author |
Abdian, Patricia Ielpi, Luis Geremía, Roberto Alejandro |
| author_facet |
Abdian, Patricia Ielpi, Luis Geremía, Roberto Alejandro |
| author_sort |
Abdian, Patricia |
| title |
Identification of essential amino acids in the bacterial α-mannosyltransferase AceA |
| title_short |
Identification of essential amino acids in the bacterial α-mannosyltransferase AceA |
| title_full |
Identification of essential amino acids in the bacterial α-mannosyltransferase AceA |
| title_fullStr |
Identification of essential amino acids in the bacterial α-mannosyltransferase AceA |
| title_full_unstemmed |
Identification of essential amino acids in the bacterial α-mannosyltransferase AceA |
| title_sort |
identification of essential amino acids in the bacterial α-mannosyltransferase acea |
| publishDate |
2000 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00219258_v275_n51_p40568_Abdian http://hdl.handle.net/20.500.12110/paper_00219258_v275_n51_p40568_Abdian |
| work_keys_str_mv |
AT abdianpatricia identificationofessentialaminoacidsinthebacterialamannosyltransferaseacea AT ielpiluis identificationofessentialaminoacidsinthebacterialamannosyltransferaseacea AT geremiarobertoalejandro identificationofessentialaminoacidsinthebacterialamannosyltransferaseacea |
| _version_ |
1768545539019767808 |