Literature summary for 2.7.8.13 extracted from

Al-Dabbagh, B.; Henry, X.; El Ghachi, M.; Auger, G.; Blanot, D.; Parquet, C.; Mengin-Lecreulx, D.; Bouhss, A.
Active site mapping of MraY, a member of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, catalyzing the first membrane step of peptidoglycan biosynthesis (2008), Biochemistry, 47, 8919-8928.

Application

Application	Comment	Organism
drug development	antibacterial chemotherapy against pathogenic bacteria being multiresistant to common antibiotics	Bacillus subtilis

Cloned(Commentary)

Cloned (Comment)	Organism
in pET28b for site-directed mutagenesis and expression with N-terminal hexa-His-tag in Escherichia coli C43(DE3) or functional complementation in BW25113-derived thermosensitive strain (EcoliTsMraY) lacking endogenous MraY	Bacillus subtilis

Protein Variants

Protein Variants	Comment	Organism
D174N	0.39% of wild-type activity, 70% of optimum activity in presence of 250 mM Mg2+, altered catalytic efficiency (kcat), not capable of functional complementation, invariant residue in third cytoplasmic segment	Bacillus subtilis
D177N	1.6% of wild-type activity, 70% of optimum activity in presence of 250 mM Mg2+, altered catalytic efficiency (kcat), invariant residue in third cytoplasmic segment	Bacillus subtilis
D231N	0.42% of wild-type activity, altered catalytic efficiency (kcat), not capable of functional complementation, invariant residue in fourth cytoplasmic segment	Bacillus subtilis
D98N	0.014% of wild-type activity, shifted pH optimum (pH 9.0-9.4) compared to wild-type (pH 7.5), highly decreased turnover (kcat, pH 7.2, elevated turnover at pH 9.4), decreased KM for UDP-N-acetylmuramoyl-pentapeptide (pH 7.2 and pH 9.4), increased KM for undecaprenyl phosphate (2fold at pH 7.2, 6fold at pH 9.4), not capable of functional complementation, invariant residue in second cytoplasmic segment	Bacillus subtilis
D99N	0.0076% of wild-type activity, highly decreased catalytic efficiency (kcat), increased KM for undecaprenyl phosphate, not capable of functional complementation, invariant residue in second cytoplasmic segment	Bacillus subtilis
E299Q	11% of wild-type activity, catalytic activity similarly affected by the presence of 250 mM Mg2+ as wild-type, slightly altered catalytic efficiency (kcat, 10%), invariant residue in fifth cytoplasmic segment	Bacillus subtilis
H289R	0.0012% of wild-type activity, highly decreased catalytic efficiency (kcat), not capable of functional complementation, invariant residue in fifth cytoplasmic segment	Bacillus subtilis
H290R	0.14% of wild-type activity, altered catalytic efficiency (kcat), not capable of functional complementation, invariant residue in fifth cytoplasmic segment	Bacillus subtilis
H291R	0.50% of wild-type activity, altered catalytic efficiency (kcat), increased KM for UDP-N-acetylmuramoyl-pentapeptide, invariant residue in fifth cytoplasmic segment	Bacillus subtilis
H45R	0.54% of wild-type activity, 70% of optimum activity in presence of 250 mM Mg2+, altered catalytic efficiency (kcat), increased KM for UDP-N-acetylmuramoyl-pentapeptide, invariant residue in first cytoplasmic segment	Bacillus subtilis
K116Q	0.046% of wild-type activity, highly decreased catalytic efficiency (kcat), not capable of functional complementation, invariant residue in second cytoplasmic segment	Bacillus subtilis
K48Q	0.51% of wild-type activity, altered catalytic efficiency (kcat), increased KM for UDP-N-acetylmuramoyl-pentapeptide, invariant residue in first cytoplasmic segment	Bacillus subtilis
N168A	1.5% of wild-type activity, altered catalytic efficiency (kcat), invariant residue in third cytoplasmic segment	Bacillus subtilis
N171A	0.34% of wild-type activity, altered catalytic efficiency (kcat), not capable of functional complementation, invariant residue in third cytoplasmic segment	Bacillus subtilis

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	undecaprenyl phosphate	3fold increase in KM for mutant D98N at pH 9.4 (ca. 1.2 mM) compared to pH 7.2	Bacillus subtilis
additional information	-	UDP-N-acetylmuramoyl-pentapeptide	no increase in KM for mutant D98N at pH 9.4 compared to pH 7.2	Bacillus subtilis
0.15	-	undecaprenyl phosphate	mutant H290R	Bacillus subtilis
0.16	-	undecaprenyl phosphate	wild-type	Bacillus subtilis
0.17	-	undecaprenyl phosphate	mutant D174N	Bacillus subtilis
0.17	-	undecaprenyl phosphate	mutant D231N	Bacillus subtilis
0.21	-	undecaprenyl phosphate	mutant N168A	Bacillus subtilis
0.22	-	undecaprenyl phosphate	mutant E299Q	Bacillus subtilis
0.22	-	undecaprenyl phosphate	mutant H45R	Bacillus subtilis
0.22	-	undecaprenyl phosphate	mutant K116Q	Bacillus subtilis
0.23	-	undecaprenyl phosphate	mutant D177N	Bacillus subtilis
0.24	-	undecaprenyl phosphate	mutant N171A	Bacillus subtilis
0.25	-	undecaprenyl phosphate	mutant H289R	Bacillus subtilis
0.26	-	undecaprenyl phosphate	mutant K48Q	Bacillus subtilis
0.27	-	undecaprenyl phosphate	mutant H291R	Bacillus subtilis
0.32	-	undecaprenyl phosphate	mutant D98N	Bacillus subtilis
0.34	-	UDP-N-acetylmuramoyl-pentapeptide	mutant D98N	Bacillus subtilis
0.35	-	undecaprenyl phosphate	mutant D99N	Bacillus subtilis
0.73	-	UDP-N-acetylmuramoyl-pentapeptide	mutant N171A	Bacillus subtilis
0.76	-	UDP-N-acetylmuramoyl-pentapeptide	mutant D99N	Bacillus subtilis
0.86	-	UDP-N-acetylmuramoyl-pentapeptide	mutant D177N	Bacillus subtilis
0.94	-	UDP-N-acetylmuramoyl-pentapeptide	wild-type	Bacillus subtilis
0.95	-	UDP-N-acetylmuramoyl-pentapeptide	mutant N168A	Bacillus subtilis
0.99	-	UDP-N-acetylmuramoyl-pentapeptide	mutant D174N	Bacillus subtilis
1	-	UDP-N-acetylmuramoyl-pentapeptide	mutant D231N	Bacillus subtilis
1.1	-	UDP-N-acetylmuramoyl-pentapeptide	mutant H289R	Bacillus subtilis
1.3	-	UDP-N-acetylmuramoyl-pentapeptide	mutant H290R	Bacillus subtilis
1.3	-	UDP-N-acetylmuramoyl-pentapeptide	mutant K48Q	Bacillus subtilis
1.4	-	UDP-N-acetylmuramoyl-pentapeptide	mutant E299Q	Bacillus subtilis
2	-	UDP-N-acetylmuramoyl-pentapeptide	mutant K116Q	Bacillus subtilis
3.4	-	UDP-N-acetylmuramoyl-pentapeptide	mutant H291R	Bacillus subtilis
3.4	-	UDP-N-acetylmuramoyl-pentapeptide	mutant H45R	Bacillus subtilis

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	optimum concentration for wild-type activity: 30-60 mM, inhibitory above 100 mM, 2% of optimum activity left at 250 mM for wild-type and 30-70% for mutants (except mutant E299Q)	Bacillus subtilis

Organism

Organism	UniProt	Comment	Textmining
Bacillus subtilis	Q03521	-	-

Purification (Commentary)

Purification (Comment)	Organism
from membrane extracts in presence of DDM detergent and by Ni-NTA metal affinity chromatography	Bacillus subtilis

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
0.00002	-	mutant H289R	Bacillus subtilis
0.00013	-	mutant D99N	Bacillus subtilis
0.00024	-	mutant D98N	Bacillus subtilis
0.00078	-	mutant K116Q	Bacillus subtilis
0.0024	-	mutant H290R	Bacillus subtilis
0.0058	-	mutant N171A	Bacillus subtilis
0.0066	-	mutant D174N	Bacillus subtilis
0.0072	-	mutant D231N	Bacillus subtilis
0.0086	-	mutant H291R	Bacillus subtilis
0.0087	-	mutant K48Q	Bacillus subtilis
0.0092	-	mutant H45R	Bacillus subtilis
0.026	-	mutant N168A	Bacillus subtilis
0.028	-	mutant D177N	Bacillus subtilis
0.18	-	mutant E299Q	Bacillus subtilis
1.7	-	wild-type	Bacillus subtilis

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	all five cytoplasmic segments contribute to catalytic site with at least 14 invariant polar and/or charged, non-clustered residues, conserved residue D98 supposedly involved in deprotonation of lipid substrate during catalysis at physiological pH 7.4 (pKa for undecaprenyl phosphate: 6.6 and 9.2)	Bacillus subtilis	?	-	?
additional information	functional complementation in a MraY-lacking, thermosensitive Escherichia coli mutant strain (2fold increased UDP-N-acetylmuramoyl-pentapeptide level compared to wild-type cells when grown 30 min at 42°C, depletion of lipid I) by overexpression of wild-type or mutants E299Q, D177N, N168A, H45R, K48Q, H291R	Bacillus subtilis	?	-	?
UDP-N-acetylmuramoyl-pentapeptide + undecaprenyl phosphate	25°C, pH 7.2	Bacillus subtilis	UMP + N-acetylmuramoyl-pentapeptide-diphosphoundecaprenol	lipid I synthesis, peptidoglycan biosynthesis	?

Synonyms

Synonyms	Comment	Organism
MraY	-	Bacillus subtilis
MraY transferase	-	Bacillus subtilis

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
additional information	-	undecaprenyl phosphate	11fold increase in kcat for mutant D98N at pH 9.4 (ca. 0.0083 1/sec) compared to pH 7.2	Bacillus subtilis
additional information	-	UDP-N-acetylmuramoyl-pentapeptide	11fold increase in kcat for mutant D98N at pH 9.4 (ca. 0.0083 1/sec) compared to pH 7.2	Bacillus subtilis
0.00005	-	undecaprenyl phosphate	mutant H289R	Bacillus subtilis
0.00005	-	UDP-N-acetylmuramoyl-pentapeptide	mutant H289R	Bacillus subtilis
0.00083	-	undecaprenyl phosphate	mutant D99N	Bacillus subtilis
0.00083	-	UDP-N-acetylmuramoyl-pentapeptide	mutant D99N	Bacillus subtilis
0.00117	-	undecaprenyl phosphate	mutant D98N	Bacillus subtilis
0.00117	-	UDP-N-acetylmuramoyl-pentapeptide	mutant D98N	Bacillus subtilis
0.0015	-	undecaprenyl phosphate	mutant K116Q	Bacillus subtilis
0.0015	-	UDP-N-acetylmuramoyl-pentapeptide	mutant K116Q	Bacillus subtilis
0.00383	-	undecaprenyl phosphate	mutant H290R	Bacillus subtilis
0.00383	-	UDP-N-acetylmuramoyl-pentapeptide	mutant H290R	Bacillus subtilis
0.0123	-	undecaprenyl phosphate	mutant D174N	Bacillus subtilis
0.0123	-	UDP-N-acetylmuramoyl-pentapeptide	mutant D174N	Bacillus subtilis
0.0135	-	undecaprenyl phosphate	mutant N171A	Bacillus subtilis
0.0135	-	UDP-N-acetylmuramoyl-pentapeptide	mutant N171A	Bacillus subtilis
0.0163	-	undecaprenyl phosphate	mutant D231N	Bacillus subtilis
0.0163	-	UDP-N-acetylmuramoyl-pentapeptide	mutant D231N	Bacillus subtilis
0.023	-	undecaprenyl phosphate	mutant H45R	Bacillus subtilis
0.023	-	UDP-N-acetylmuramoyl-pentapeptide	mutant H45R	Bacillus subtilis
0.027	-	undecaprenyl phosphate	mutant K48Q	Bacillus subtilis
0.027	-	UDP-N-acetylmuramoyl-pentapeptide	mutant K48Q	Bacillus subtilis
0.0317	-	undecaprenyl phosphate	mutant H291R	Bacillus subtilis
0.0317	-	UDP-N-acetylmuramoyl-pentapeptide	mutant H291R	Bacillus subtilis
0.065	-	undecaprenyl phosphate	mutant D177N	Bacillus subtilis
0.065	-	UDP-N-acetylmuramoyl-pentapeptide	mutant D177N	Bacillus subtilis
0.0717	-	undecaprenyl phosphate	mutant N168A	Bacillus subtilis
0.0717	-	UDP-N-acetylmuramoyl-pentapeptide	mutant N168A	Bacillus subtilis
0.53	-	undecaprenyl phosphate	mutant E299Q	Bacillus subtilis
0.53	-	UDP-N-acetylmuramoyl-pentapeptide	mutant E299Q	Bacillus subtilis
5.3	-	undecaprenyl phosphate	wild-type	Bacillus subtilis
5.3	-	UDP-N-acetylmuramoyl-pentapeptide	wild-type	Bacillus subtilis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	wild-type and all mutants except mutant D98N	Bacillus subtilis
9.4	-	9.0-9.4, mutant D98N	Bacillus subtilis

pH Range

pH Minimum	pH Maximum	Comment	Organism
6.8	9.4	mutant D98N shows different pH profile (specific activity ratio between pH9.4/pH7.6: 2.5) than wild-type (and all mutants except mutant D98N, specific activity ratio between pH9.4/pH7.6: 0.22-0.83)	Bacillus subtilis