Information on EC 2.3.2.17 - N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysyl-(N6-glycyl)-D-alanyl-D-alanine-diphosphoundecaprenyl-N-acetylglucosamine:glycine glycyltransferase

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The expected taxonomic range for this enzyme is: Staphylococcus

EC NUMBER
COMMENTARY
2.3.2.17
-
RECOMMENDED NAME
GeneOntology No.
N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysyl-(N6-glycyl)-D-alanyl-D-alanine-diphosphoundecaprenyl-N-acetylglucosamine:glycine glycyltransferase
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
MurNAc-L-Ala-D-isoglutaminyl-L-Lys-(N6-Gly)-D-Ala-D-Ala-diphospho-ditrans,octacis-undecaprenyl-GlcNAc + 2 glycyl-tRNAGly = MurNAc-L-Ala-D-isoglutaminyl-L-Lys-(N6-tri-Gly)-D-Ala-D-Ala-diphospho-ditrans,octacis-undecaprenyl-GlcNAc + 2 tRNAGly
show the reaction diagram
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
Metabolic pathways
-
Peptidoglycan biosynthesis
-
peptidoglycan cross-bridge biosynthesis I (S. aureus)
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SYSTEMATIC NAME
IUBMB Comments
N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysyl-(N6-glycyl)-D-alanyl-D-alanine-diphosphoundecaprenyl-N-acetylglucosamine:glycine glycyltransferase
This enzyme catalyses the successive transfer of two Gly moieties from charged tRNAs to MurNAc-L-Ala-D-isoglutaminyl-L-Lys-(N6-Gly)-D-Ala-D-Ala-diphospho-ditrans,octacis-undecaprenyl-GlcNAc, attaching them to a Gly residue previously attached by EC 2.3.2.16 (lipid II:glycine glycyltransferase) to the N6 of the L-Lys at position 3 of the pentapeptide. This is the second step in the synthesis of the pentaglycine interpeptide bridge that is used by Staphylococcus aureus for the crosslinking of different glycan strands to each other. The next step is catalysed by EC 2.3.2.18 (N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysyl-(N6-triglycine)-D-alanyl-D-alanine-diphosphoundecaprenyl-N-acetylglucosamine:glycine glycyltransferase). This enzyme is essential for methicillin resistance [1].
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
FemA
D4N2Z4
gene name
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
clinical isolates MRSA252, SA 1306, SA 1326, SA 1552 and SA 4666
D4N2Z4
UniProt
Manually annotated by BRENDA team
methicillin-resistant strain
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
malfunction
D4N2Z4
decreased expression of the femA gene leads to reduced methicillin resistance
metabolism
-
all investigated strains, either methicillin-resistant or susceptible, express FemA during the exponential growth phase in varying amounts. In the stationary phase, the FemA content is diminished. Strains in which FemA is inactivated by insertion of Tn551 into the control region of the FemAB operon still express about 10% of the protein compared to their parent strains. Tn551 insertion in the middle of the femB gene does not affect the FemA expression
physiological function
-
femA mutants leading to truncated proteins still produce intact FemB while exhibiting a phenotype identical to femAB double mutants, such as same muropeptide pattern. FemA is essential for the addition of glycine residues 2 and 3 only to the staphylococcal interpeptide bridge
physiological function
-
surface protein is linked to tri- and monoglycyl cross-bridges of peptidoglycan isolated from femB and femA mutant staphylococci, respectively. No surface protein is found linked directly to the epsilon-amino group of lysyl within the cell wall of a femAX strain. Peptidoglycan analysis of a femAB mutant strain reveals the presence of pentaglycyl, tetraglycyl-monoseryl, and monoglycyl as well as small amounts of triglycyl cross-bridges. Analysis of anchor peptides shows that surface proteins are mostly linked to tetraglycylmonoseryl as well as pentaglycyl. The sortase activity of Staphylococcus aureus prefers cross-bridges containing five residues, but altered cell-wall cross-bridges can be linked to the COOH-terminal end of surface proteins
physiological function
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expression levels of femA in methicillin-sensitive, low-level methicillin-resistant and high-level methicillin-resistant Staphylococcus aureus are 0.035%-29.91%, 0.055%-310% and 13.88-5500%, respectively. EMSA detects a signal shift in 57 high-level methicillin-resistant isolates but not in four low-level methicillin-resistant and four methicillin-sensitive strains. Expression of femA in high-level methicillin-resistant non-beta-lactamase-producing strains is higher than in low-level methicilln-resistant and methicillin-sensitive strains
physiological function
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FemA is needed for cell growth in the presence of beta-lactam antibiotics, it has no influence on the synthesis of the low affinity, additional penicillin-binding protein PBP2' encoded by mec and known to be essential for cell wall synthesis in the presence of inhibitory concentrations of methicillin
physiological function
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FemA catalyzes the second step in the synthesis of the pentaglycine interpeptide bridge crosslinking different glycan strands in Staphylococcus aureus. FemX adds the first glycine residue to MurNAc-L-Ala-D-Glu-(N6-Gly)L-Lys-D-Ala-D-Ala-diphosphoundecaprenyl-M-acetylglucosamine, i.e. lipid II. Addition of glycine residues 2, 3 and glycine residues 4, 5 is catalyzed by enzymes FemA and FemB, respectively. None of the FemABX enzymes requires the presence of one or two of the other Fem proteins for activity, rather, bridge formation is delayed in an in vitro system when all 3 enzymes are present
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
N-acetylmuramoyl-L-alanyl-D-isoglutaminyl-L-lysyl-(N6-glycyl)-D-alanyl-D-alanine-diphosphoundecaprenyl-N-acetylglucosamine + 2 glycyl-tRNA
N-acetylmuramoyl-L-alanyl-D-isoglutaminyl-L-lysyl-(N6-triglycyl)-D-alanyl-D-alanine-diphosphoundecaprenyl-N-acetylglucosamine + 2 tRNA
show the reaction diagram
-
i.e. lipid II-Gly. Enzyme is specific for lipid II-Gly as acceptor
-
-
?
pI VALUE
pI VALUE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
8.5
-
P95734
calculated
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
89300
-
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
P95734
x * 49564, calculated
?
-
x *48000, SDS-PAGE, x * 50600, calculated
?
-
x * 49369, calculated
?
-
x * 49092, calculated
dimer
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2 * 49400, SDS-PAGE
additional information
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proteins FemA and FemB form homo- and heterodimers in vitro
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
to 2.1 A resolution. The FemA structure reveals a unique organization of several known protein folds involved in peptide and tRNA binding. The surface of the protein reveals an L-shaped channel suitable for a peptidoglycan substrate
P0A0A5
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expression as His-tagged protein in Escherichia coli
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expression in Escherichia coli
P95734
EXPRESSION
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
femA gene expression is not upregulated in oxacillin susceptible methicillin-resistant OS-MRSA strains compared to low- and high-level methicillin-resistant MRSA control strains
D4N2Z4
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
the lysostaphin immunity factor Lif is not able to complement lack of FemA by inserting serine for glycine in the side chain. Methicillin resistance, which depends on functional FemA and FemB, is neither complemented by Lif
additional information
-
analysis of several related methicillin-resistant, methicillin-susceptible, and TnS51 insertionally inactivated femA mutants. All mutants have a reduced peptidoglycan glycine content compared to that of related femA parent strains. Additional effects of femA inactivation and the subsequent decrease in peptidoglycan-associated glycine are reduced digestion of peptidoglycan by recombinant lysostaphin, unaltered digestion of peptidoglycan by Chalaropsis B-muramidase, reduced cell wall turnover, reduced whole-cell autolysis, and increased sensitivity towards beta-lactam antibiotics. The peptidoglycan-associated glycine content of a femA::Tn5Sl methicillin-susceptible strain is restored concomitantly with the methicillin resistance to a level almost equal to that of its femA4 methicillin-resistant parent strain by introduction of a plasmid encoding femA
additional information
-
in strains carrying mutations of FemA, femAB, or the femAX genes, the sorting reaction of surface proteins is significantly slowed. Strains carrying mutations in the fem genes display a decreased rate of surface protein precursor cleavage as compared with the wildtype strains, suggesting that the altered cross-bridges slow the anchoring of surface proteins
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
medicine
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in 40 methicillin-susceptible and 6 resistant clinical isolates of Staphylococcus aureus, the FemA content or its affinity to the antibodies is reduced compared to laboratory parent strains. In susceptible strains, an additional protein of higher molecular weight, present in large quantities, is also able to bind the FemA antibodies. Such a protein is also present in methicillin-resistant isolates, although it is not as pronounced as in the susceptible strains
medicine
-
expression levels of femA in methicillin-sensitive, low-level methicillin-resistant and high-level methicillin-resistant Staphylococcus aureus are 0.035%-29.91%, 0.055%-310% and 13.88-5500%, respectively. EMSA detects a signal shift in 57 high-level methicillin-resistant isolates but not in four low-level methicillin-resistant and four methicillin-sensitive strains. Expression of femA in high-level methicillin-resistant non-beta-lactamase-producing strains is higher than in low-level methicilln-resistant and methicillin-sensitive strains
analysis
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definition of a consensus sequence of the femA gene and analysis of interspecies variations for staphylococci species-specific identification