Literature summary for 5.1.3.14 extracted from

Mann, P.A.; Mueller, A.; Wolff, K.A.; Fischmann, T.; Wang, H.; Reed, P.; Hou, Y.; Li, W.; Mueller, C.E.; Xiao, J.; Murgolo, N.; Sher, X.; Mayhood, T.; Sheth, P.R.; Mirza, A.; Labroli, M.; Xiao, L.; McCoy, M.; Gill, C.J.; Pinho, M.G.; Schneider, T.; Roemer, T.
Chemical genetic analysis and functional characterization of staphylococcal wall teichoic acid 2-epimerases reveals unconventional antibiotic drug targets (2016), PLoS Pathog., 12, e1005585 .

Application

Application	Comment	Organism
drug development	Staphylococcus aureus is a leading cause of hospital and community-acquired infections by Gram-positive bacteria and Staphylococcus epidermidis has emerged as the most common cause of biofilm infections on medical implant devices. Enzyme MnaA serves as a Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore beta-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation	Staphylococcus epidermidis
drug development	Staphylococcus aureus is a leading cause of hospital and community-acquired infections by Gram-positive bacteria and Staphylococcus epidermidis has emerged as the most common cause of biofilm infections on medical implant devices. Enzyme MnaA serves as a Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore beta-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation	Staphylococcus aureus

Cloned(Commentary)

Cloned (Comment)	Organism
gene mnaA, genotyping and mutation identification	Staphylococcus epidermidis
gene mnaA, genotyping and mutation identification	Staphylococcus aureus

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified enzyme MnaA, hanging drop vapor diffusion method, mixing of 31 mg/ml protein solution with crystallization solution containing 0.1 M Tris-HCl, pH 8.0, 0.1 M Na2SO4, 52% PEG 400, 22°C, method optimization, X-ray diffraction structure determination and analysis at 1.9 A resolution, molecular replacement using structure PDB ID 1F6D as template	Staphylococcus aureus

Protein Variants

Protein Variants	Comment	Organism
additional information	gene mnaA, genotyping and mutation identification. Mapping of MnaA LOF mutations into the MnaA crystal structure revealing key residues for substrate binding site stability and charge. To determine whether L638R mnaA LOF mutants are not identified in MRSA COL due to a functional redundancy between Cap5P and MnaA, a cap5P deletion mutant is constructed and the L638R studies are repeated. Under these conditions, in addition to identifying the expected tarG L638R mutations as well as tarO and tarA LOF mutations, multiple (n = 11) independent resistor isolates obtained uniquely possess distinct mutations that map to mnaA and are predicted to inactivate gene function as well as directly confer L638R drug resistance based on the absence of additional non-synonymous mutations in their genome following WGS analysis. While MRSA COL DELTAcap5P exhibits no wall teichoic acid (WTA) depletion phenotype and remains resistant to beta-lactams, MRSA COL mnaA, DELTAcap5P double mutants are completely devoid of WTA and are also highly sensitive to beta-lactams. MRSA COL mnaA, cap5P double mutants and MRSE mnaA single mutants reveal morphological phenotypes consistent with WTA depletion, including increased cell size heterogeneity and septation defects. Complementing DELTAcap5P mnaASa P12L and DELTAcap5P mnaASa Y194* with either cap5P or mnaASa reintroduced on an inducible plasmid restores WTA polymer levels, resistance to each of the beta-lactams tested, and wild-type sensitivity to L638	Staphylococcus aureus
additional information	gene mnaA, genotyping and mutation identification. MRSA COL mnaA, cap5P double mutants and MRSE mnaA single mutants reveal morphological phenotypes consistent with WTA depletion, including increased cell size heterogeneity and septation defects	Staphylococcus epidermidis
additional information	to determine whether L638R mnaA LOF mutants are not identified in MRSA COL due to a functional redundancy between Cap5P and MnaA, a cap5P deletion mutant is constructed and the L638R studies are repeated. Under these conditions, in addition to identifying the expected tarG L638R mutations as well as tarO and tarA LOF mutations, multiple (n = 11) independent resistor isolates obtained uniquely possess distinct mutations that map to mnaA and are predicted to inactivate gene function as well as directly confer L638R drug resistance based on the absence of additional non-synonymous mutations in their genome following WGS analysis. While MRSA COL DELTAcap5P exhibits no wall teichoic acid (WTA) depletion phenotype and remains resistant to beta-lactams, MRSA COL mnaA, DELTAcap5P double mutants are completely devoid of WTA and are also highly sensitive to beta-lactams. MRSA COL mnaA, cap5P double mutants and MRSE mnaA single mutants reveal morphological phenotypes consistent with WTA depletion, including increased cell size heterogeneity and septation defects	Staphylococcus aureus
P12L	site-diected mutagenesis	Staphylococcus aureus
Y194X	site-directed mutagenesis	Staphylococcus aureus

Inhibitors

Inhibitors	Comment	Organism
additional information	WTA 2-epimerases are dual beta-lactam potentiation and antibiofilm drug targets; WTA 2-epimerases are dual beta-lactam potentiation and antibiofilm drug targets	Staphylococcus aureus
additional information	WTA 2-epimerases are dual beta-lactam potentiation and antibiofilm drug targets	Staphylococcus epidermidis
tunicamycin	the natural product antibiotic physically binds Cap5P and inhibits 2-epimerase activity, NMR study, reversible inhibition; the natural product antibiotic physically binds MnaA and inhibits 2-epimerase activity, NMR study, reversible inhibition	Staphylococcus aureus
tunicamycin	the natural product antibiotic physically binds MnaA and inhibits 2-epimerase activity, NMR study, reversible inhibition	Staphylococcus epidermidis

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
cell wall	-	Staphylococcus epidermidis	5618	-
cell wall	-	Staphylococcus aureus	5618	-

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
UDP-N-acetyl-alpha-D-glucosamine	Staphylococcus epidermidis	-	UDP-N-acetyl-alpha-D-mannosamine	-	r
UDP-N-acetyl-alpha-D-glucosamine	Staphylococcus aureus	-	UDP-N-acetyl-alpha-D-mannosamine	-	r
UDP-N-acetyl-alpha-D-glucosamine	Staphylococcus aureus COL	-	UDP-N-acetyl-alpha-D-mannosamine	-	r
UDP-N-acetyl-alpha-D-glucosamine	Staphylococcus epidermidis CLB26329	-	UDP-N-acetyl-alpha-D-mannosamine	-	r

Organism

Organism	UniProt	Comment	Textmining
Staphylococcus aureus	P95709	MRSA	-
Staphylococcus aureus	Q9REV4	MRSA	-
Staphylococcus aureus COL	P95709	MRSA	-
Staphylococcus aureus COL	Q9REV4	MRSA	-
Staphylococcus epidermidis	-	MRSE	-
Staphylococcus epidermidis CLB26329	-	MRSE	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
UDP-N-acetyl-alpha-D-glucosamine	-	Staphylococcus epidermidis	UDP-N-acetyl-alpha-D-mannosamine	-	r
UDP-N-acetyl-alpha-D-glucosamine	-	Staphylococcus aureus	UDP-N-acetyl-alpha-D-mannosamine	-	r
UDP-N-acetyl-alpha-D-glucosamine	interconversion	Staphylococcus epidermidis	UDP-N-acetyl-alpha-D-mannosamine	-	r
UDP-N-acetyl-alpha-D-glucosamine	interconversion	Staphylococcus aureus	UDP-N-acetyl-alpha-D-mannosamine	-	r
UDP-N-acetyl-alpha-D-glucosamine	-	Staphylococcus aureus COL	UDP-N-acetyl-alpha-D-mannosamine	-	r
UDP-N-acetyl-alpha-D-glucosamine	interconversion	Staphylococcus aureus COL	UDP-N-acetyl-alpha-D-mannosamine	-	r
UDP-N-acetyl-alpha-D-glucosamine	-	Staphylococcus epidermidis CLB26329	UDP-N-acetyl-alpha-D-mannosamine	-	r
UDP-N-acetyl-alpha-D-glucosamine	interconversion	Staphylococcus epidermidis CLB26329	UDP-N-acetyl-alpha-D-mannosamine	-	r
UDP-N-acetyl-alpha-D-mannosamine	interconversion	Staphylococcus epidermidis	UDP-N-acetyl-alpha-D-glucosamine	-	r
UDP-N-acetyl-alpha-D-mannosamine	interconversion	Staphylococcus aureus	UDP-N-acetyl-alpha-D-glucosamine	-	r
UDP-N-acetyl-alpha-D-mannosamine	interconversion	Staphylococcus aureus COL	UDP-N-acetyl-alpha-D-glucosamine	-	r
UDP-N-acetyl-alpha-D-mannosamine	interconversion	Staphylococcus epidermidis CLB26329	UDP-N-acetyl-alpha-D-glucosamine	-	r

Subunits

Subunits	Comment	Organism
dimer	-	Staphylococcus aureus

Synonyms

Synonyms	Comment	Organism
Cap5P	-	Staphylococcus aureus
MnaA	-	Staphylococcus epidermidis
MnaA	-	Staphylococcus aureus
teichoic acid 2-epimerase	-	Staphylococcus epidermidis
teichoic acid 2-epimerase	-	Staphylococcus aureus
UDP-GlcNAc:UDP-ManNAc 2-epimerase	-	Staphylococcus epidermidis
UDP-GlcNAc:UDP-ManNAc 2-epimerase	-	Staphylococcus aureus
WTA 2-epimerase	-	Staphylococcus epidermidis
WTA 2-epimerase	-	Staphylococcus aureus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Staphylococcus epidermidis
30	-	assay at	Staphylococcus aureus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Staphylococcus epidermidis
8	-	assay at	Staphylococcus aureus

General Information

General Information	Comment	Organism
malfunction	deletions of early wall teichoic acid (WTA) biosynthetic enzymes are nonlethal, but cause diverse attenuated virulence phenotypes, deletions of later steps in WTA biosynthesis are not generally tolerated and the enzymes are normally essential for growth, an essential gene paradox. The beta-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA infection. Complementing DELTAcap5P mnaASa P12L and DELTAcap5P mnaASa Y194* with either cap5P or mnaASa reintroduced on an inducible plasmid restores WTA polymer levels, resistance to each of the beta-lactams tested, and wild-type sensitivity to L638	Staphylococcus aureus
malfunction	deletions of early wall teichoic acid (WTA) biosynthetic enzymes are nonlethal, but cause diverse attenuated virulence phenotypes, deletions of later steps in WTA biosynthesis are not generally tolerated and the enzymes are normally essential for growth, an essential gene paradox. The beta-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSE infection	Staphylococcus epidermidis
physiological function	2-epimerase MnaA interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Besides MnaA, Staphylococcus aureus maintains a second 2-epimerase involved in serotype 5 capsular polysaccharide (CP5) synthesis, Cap5P. MnaA and Cap5P provide compensatory WTA functional roles in Staphylococcus aureus. MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA. Overlapping functional activity of MnaA and Cap5P in Staphylococci	Staphylococcus aureus
physiological function	2-epimerase MnaA interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. MnaA serves as the sole 2-epimerase required for WTA biosynthesis in Staphylococcus epidermidis. MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSE	Staphylococcus epidermidis