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Enzyme Nomenclature
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 and Organism(s) Staphylococcus aureus and UniProt Accession P0A0A5
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2.3.2.17 N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysyl-(N6-glycyl)-D-alanyl-D-alanine-diphosphoundecaprenyl-N-acetylglucosamine:glycine glycyltransferaseIUBMB Comments
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 .
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The taxonomic range for the selected organisms is: Staphylococcus aureus
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
FemA
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].
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
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
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i.e. lipid II-Gly. Enzyme is specific for lipid II-Gly as acceptor
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-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
malfunction
a femA deletion leads to accumulation of monoglycine which decreases the interpeptide cross-linking in the peptidoglycan (PGN) sacculus as compared to wild-type cells
metabolism
kinase Stk and phosphatase Stp modulate cell wall synthesis and cell division at several levels. Enzyme FemA interacts with the eukaryotic-like serine/threonine kinase Stk, but is not phosphorylated by it, while the lipid II:glycine glycyltransferase FemX can be phosphorylated by the Ser/Thr kinase Stk in vitro. The cognate phosphatase Stp dephosphorylates these phosphorylation sites. Stk interacts with FemA/B and other cell wall synthesis and cell division proteins, but Stk does not phosphorylate FemA and FemB. Interaction network of Stk, Stp and FemX/A/B proteins among cell wall synthesis and cell division proteins as determined by bacterial two-hybrid analysis, overview
physiological function
the bacterial cell envelope is essential for survival and pathogenicity. It forms a barrier against environmental stresses and contributes to virulence and antibiotic resistance. The cell wall of Gram-positive bacteria is composed of a multi-layered mesh of cross-linked peptidoglycan (PGN). PGN consists of chains of repeating disaccharide units comprising N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). The lactoyl group of MurNAc is supplemented with a penta stem peptide (L-Ala-D-isoGlu-L-Lys-D-Ala-D-Ala). The staphylococcal PGN polysaccharide chains are highly cross-linked via interpeptide bridges of five glycyl residues protruding from the L-lysine of the stem-peptides4. These interpeptide bridges are synthesized by the FemX/A/B enzymes. These non-ribosomal peptidyl-transferases use glycyltRNAs to sequentially add five glycine's to the PGN-lysyl side chain of lipid II. FemX adds the first glycyl unit, FemA the second and third unit, and FemB adds the fourth and fifth glycyl unit to complete the pentaglycine-bridge. Enzyme FemA interacts with the eukaryotic-like serine/threonine kinase Stk, but is not phosphorylated by it. FemA and FemB interact with Stk and with cell wall synthesis enzymes (MurG, Pbp1, Pbp2), Mgt, LytH, RodA, FtsW and cell division proteins (DivIB, DivIC, EzrA). FemA and FemB interact with each other and also form homodimers, which is not the case for FemX. In contrast to FemX, the subsequent enzymes FemA or FemB are non-essential
malfunction
decreased expression of the femA gene leads to reduced methicillin resistance
metabolism
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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
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
-
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
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 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
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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
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
proteins FemA and FemB form homo- and heterodimers in vitro
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
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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
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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
additional information
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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
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant N-terminally polyHis-tagged enzyme FemA from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, dialysis, and ultrafiltration
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene femA, recombinant expression of N-terminally polyHis-tagged enzyme FemA in Escherichia coli strain BL21(DE3)
EXPRESSION
ORGANISM
UNIPROT
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
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
medicine
medicine
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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
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
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Johnson, S.; Kruger, D.; Labischinski, H.
FemA of Staphylococcus aureus: isolation and immunodetection
FEMS Microbiol. Lett.
132
221-228
1995
Staphylococcus aureus
Tschierske, M.; Ehlert, K.; Stranden, A.M.; Berger-Bachi, B.
Lif, the lysostaphin immunity factor, complements FemB in staphylococcal peptidoglycan interpeptide bridge formation
FEMS Microbiol. Lett.
153
261-264
1997
Staphylococcus aureus
Maidhof, H.; Reinicke, B.; Blumel, P.; Berger-Bachi, B.; Labischinski, H.
femA, Which encodes a factor essential for expression of methicillin resistance, affects glycine content of peptidoglycan in methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains
J. Bacteriol.
173
3507-3513
1991
Staphylococcus aureus
Ehlert, K.; Schroder, W.; Labischinski, H.
Specificities of FemA and FemB for different glycine residues: FemB cannot substitute for FemA in staphylococcal peptidoglycan pentaglycine side chain formation
J. Bacteriol.
179
7573-7576
1997
Staphylococcus aureus
Ton-That, H.; Labischinski, H.; Berger-Bachi, B.; Schneewind, O.
Anchor structure of staphylococcal surface proteins. III. Role of the FemA, FemB, and FemX factors in anchoring surface proteins to the bacterial cell wall
J. Biol. Chem.
273
29143-29149
1998
Staphylococcus aureus
Li, X.; Xiong, Y.; Fan, X.; Zhong, Z.; Feng, P.; Tang, H.; Zhou, T.
A study of the regulating gene of femA from methicillin-resistant Staphylococcus aureus clinical isolates
J. Int. Med. Res.
36
420-433
2008
Staphylococcus aureus
Rohrer, S.; Berger-Bachi, B.
Application of a bacterial two-hybrid system for the analysis of protein-protein interactions between FemABX family proteins
Microbiology
149
2733-2738
2003
Staphylococcus aureus
Berger-Bachi, B.; Barberis-Maino, L.; Strassle, A.; Kayser, F.H.
FemA, a host-mediated factor essential for methicillin resistance in Staphylococcus aureus: molecular cloning and characterization
Mol. Gen. Genet.
219
263-269
1989
Staphylococcus aureus
Schneider, T.; Senn, M.M.; Berger-Bachi, B.; Tossi, A.; Sahl, H.G.; Wiedemann, I.
In vitro assembly of a complete, pentaglycine interpeptide bridge containing cell wall precursor (lipid II-Gly5) of Staphylococcus aureus
Mol. Microbiol.
53
675-685
2004
Staphylococcus aureus
Benson, T.E.; Prince, D.B.; Mutchler, V.T.; Curry, K.A.; Ho, A.M.; Sarver, R.W.; Hagadorn, J.C.; Choi, G.H.; Garlick, R.L.
X-ray crystal structure of Staphylococcus aureus FemA
Structure
10
1107-1115
2002
Staphylococcus aureus (P0A0A5)
Giannouli, S.; Labrou, M.; Kyritsis, A.; Ikonomidis, A.; Pournaras, S.; Stathopoulos, C.; Tsakris, A.
Detection of mutations in the FemXAB protein family in oxacillin-susceptible mecA-positive Staphylococcus aureus clinical isolates
J. Antimicrob. Chemother.
65
626-633
2010
Staphylococcus aureus (D4N2Z4)
Jarick, M.; Bertsche, U.; Stahl, M.; Schultz, D.; Methling, K.; Lalk, M.; Stigloher, C.; Steger, M.; Schlosser, A.; Ohlsen, K.
The serine/threonine kinase Stk and the phosphatase Stp regulate cell wall synthesis in Staphylococcus aureus
Sci. Rep.
8
13693
2018
Staphylococcus aureus (P0A0A5)
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