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C115D
the mutant enzyme lacks the ability to react with phosphoenolpyruvate covalently
C251S
-
site-directed mutagenesis, Cys251 is not involved in the catalysis, unaltered biochemical properties
C354S
-
site-directed mutagenesis, Cys354 is not involved in the catalysis, unaltered biochemical properties
C381S
-
site-directed mutagenesis, Cys381 is not involved in the catalysis, unaltered biochemical properties
D305C
-
site-directed mutagenesis, weaker binding of UDP-GlcNAc, no activity
D305E
-
site-directed mutagenesis, weaker binding of UDP-GlcNAc, 0.1% activity compared to the wild-type
D305H
-
site-directed mutagenesis, weaker binding of UDP-GlcNAc, no activity, fosfomycin is not covalently attached to Cys115
K22V/R120K
no residual activity, heat capacity changes are markedly redcued
K22V/R120V
no residual activity
N23A
-
site-directed mutagenesis, reduced activity, 20fold higher apparent dissociation constant for fosfomycin compared to wild-type
N23S
-
site-directed mutagenesis, reduced activity, 200fold higher apparent dissociation constant for fosfomycin compared to wild-type
R120K
less than 0.05% of wild-type activity, heat capacity changes are markedly redcued
C251S
-
site-directed mutagenesis, Cys251 is not involved in the catalysis, unaltered biochemical properties
-
C354S
-
site-directed mutagenesis, Cys354 is not involved in the catalysis, unaltered biochemical properties
-
C381S
-
site-directed mutagenesis, Cys381 is not involved in the catalysis, unaltered biochemical properties
-
D305A
-
site-directed mutagenesis, weaker binding of UDP-GlcNAc, no activity, fosfomycin is not covalently attached to Cys115
-
D305C
-
site-directed mutagenesis, weaker binding of UDP-GlcNAc, no activity
-
D305E
-
site-directed mutagenesis, weaker binding of UDP-GlcNAc, 0.1% activity compared to the wild-type
-
K22E
-
site-directed mutagenesis, exchange of conserved Lys residue located near the active site and involved in substrate binding leading to conformational changes, shows less than 0.5% activity compared to the wild-type, altered UDP-GlcNAc binding, highly reduced formation of covalent adduct between active site Cys115 and phosphoenolpyruvate or inhibitor fosfomycin
-
K22R
-
site-directed mutagenesis, exchange of conserved Lys residue located near the active site and involved in substrate binding leading to conformational changes, shows less than 0.5% activity compared to the wild-type, slightly reduced formation of covalent adduct between active site Cys115 and phosphoenolpyruvate or inhibitor fosfomycin
-
K22V
-
site-directed mutagenesis, exchange of conserved Lys residue located near the active site and involved in substrate binding leading to conformational changes, shows less than 0.5% activity compared to the wild-type, reduced formation of covalent adduct between active site Cys115 and phosphoenolpyruvate or inhibitor fosfomycin
-
N23A
-
site-directed mutagenesis, reduced activity, 20fold higher apparent dissociation constant for fosfomycin compared to wild-type
-
N23S
-
site-directed mutagenesis, reduced activity, 200fold higher apparent dissociation constant for fosfomycin compared to wild-type
-
C120S
-
catalytically inactive
C115A
-
site-directed mutagenesis, overexpression in Escherichia coli, no activity
C115E
-
site-directed mutagenesis, overexpression in Escherichia coli, gains fosfomycin resistance, enhanced pH-dependency of the reaction, low activity
C115N
-
site-directed mutagenesis, overexpression in Escherichia coli, deamination of Asn115 to Asp115
C115S
-
site-directed mutagenesis, overexpression in Escherichia coli, no activity
C115A
-
site-directed mutagenesis, overexpression in Escherichia coli, no activity
-
C115D
-
site-directed mutagenesis, overexpression in Escherichia coli, gains fosfomycin resistance, forms only the phospholactyl-enzyme intermediate adduct, but no UDP-GlcNAc-phosphoenolpyruvate, higher kcat than the wild-type at pH 7.0, enhanced pH-dependency of the reaction
-
C115E
-
site-directed mutagenesis, overexpression in Escherichia coli, gains fosfomycin resistance, enhanced pH-dependency of the reaction, low activity
-
C115N
-
site-directed mutagenesis, overexpression in Escherichia coli, deamination of Asn115 to Asp115
-
C115S
-
site-directed mutagenesis, overexpression in Escherichia coli, no activity
-
C115D
-
mutant protein is functional and resistant to fosfomycin and inhibitors 2-[4-(2-hydroxyethyl)piperazin-1-yl]-6,7-dimethoxy-3,4-dihydronaphthalen-1(2H)-one and2-(4-methylpiperazin-1-yl)-3,4-dihydronaphthalen-1(2H)-one
D120C
the mutant shows resistance against fosfomycin
C115S
-
-
C115S
mutant enzyme is capable of catalyzing the wild type enzyme reaction but is unable to release the products (single turnover), activity is not affected by fosfomycin
D305A
-
site-directed mutagenesis, weaker binding of UDP-GlcNAc, no activity, fosfomycin is not covalently attached to Cys115
D305A
-
crystallization data
K22E
site-directed mutagenesis, exchange of conserved Lys residue located near the active site and involved in substrate binding leading to conformational changes, shows less than 0.5% activity compared to the wild-type, altered UDP-GlcNAc binding, highly reduced formation of covalent adduct between active site Cys115 and phosphoenolpyruvate or inhibitor fosfomycin
K22E
0.05% of wild-type activity, no formation of covalent adduct with fosfomycin
K22R
0.3% of wild-type activity
K22R
site-directed mutagenesis, exchange of conserved Lys residue located near the active site and involved in substrate binding leading to conformational changes, shows less than 0.5% activity compared to the wild-type, slightly reduced formation of covalent adduct between active site Cys115 and phosphoenolpyruvate or inhibitor fosfomycin
K22V
site-directed mutagenesis, exchange of conserved Lys residue located near the active site and involved in substrate binding leading to conformational changes, shows less than 0.5% activity compared to the wild-type, reduced formation of covalent adduct between active site Cys115 and phosphoenolpyruvate or inhibitor fosfomycin
K22V
0.03% of wild-type activity, similar to wild-type in binding of fosfomycin, presence of UDP-N-acetylglucosamine required
C115D
-
site-directed mutagenesis, overexpression in Escherichia coli, gains fosfomycin resistance, forms only the phospholactyl-enzyme intermediate adduct, but no UDP-GlcNAc-phosphoenolpyruvate, higher kcat than the wild-type at pH 7.0, enhanced pH-dependency of the reaction
C115D
mutant protein is functional and resistant to fosfomycin and inhibitors 2-[4-(2-hydroxyethyl)piperazin-1-yl]-6,7-dimethoxy-3,4-dihydronaphthalen-1(2H)-one and2-(4-methylpiperazin-1-yl)-3,4-dihydronaphthalen-1(2H)-one
C117D
-
the mutation shifts ithe optimum pH from 7.8 to 6.0. The mutant is not inhibited by 1 mM fosfomycin
C117D
-
the mutant is not inhibited by fosfomycin
additional information
-
antisense-based modulation of murA1 gene expression, which encodes UDP-N-acetylglucosamine enolpyruvyl transferase, hypersensitizes cells to the MurA-specific antibiotic fosfomycin despite the normally high resistance of Bacillus anthracis to this drug
additional information
construction of a murA(Z) deletion mutant strain without catalytic activity, cells grow only when complemented by the enzyme expressed from a plasmid
additional information
-
construction of a murA(Z) deletion mutant strain without catalytic activity, cells grow only when complemented by the enzyme expressed from a plasmid
additional information
-
individually inactivation of the 2 isoforms by allelic replacement shows, that the 2 forms can substitute for each other, a double deletion mutant is not viable