Staphylococcus aureus MurC participates in L-alanine recognition via histidine 343, a conserved motif in the shallow hydrophobic pocket.
UDP-N-acetylmuramic acid:L-alanine ligase that is encoded by the murC gene, is indispensable for bacterial peptidoglycan biosynthesis and an important target for the development of antibacterial agents. Structure of MurC ligase with substrates has been described, however, little validation via studying the effects of mutations on the structure of MurC has been performed. In this study, we carried out a functional in vitro and in vivo characterization of Staphylococcus aureus MurCH343Y protein that has a temperature-sensitive mutation of a conserved residue in the predicted shallow hydrophobic pocket that holds a short L-alanine side chain. Purified H343Y and wild-type MurC had K(m) values for L-alanine of 3.2 and 0.44 mM, respectively, whereas there was no significant difference in their K(m) values for ATP and UDP-N-acetylmuramic acid, suggesting the specific alteration of L-alanine recognition in MurCH343Y protein. In a synthetic medium that excluded L-alanine, S. aureus murCH343Y mutant cells showed an allele-specific slow growth phenotype that was suppressed by addition of L-alanine. These results suggest that His343 of S. aureus MurC is essential for high-affinity binding to L-alanine both in vitro and in vivo and provide experimental evidence supporting the structural information of MurC ligase.
Kurokawa
Kenji
K
Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan. kurokawa@mol.f.u-tokyo.ac.jp
Nishida
Satoshi
S
Ishibashi
Mihoko
M
Mizumura
Hikaru
H
Ueno
Kohji
K
Yutsudo
Takashi
T
Maki
Hideki
H
Murakami
Kazuhisa
K
Sekimizu
Kazuhisa
K
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000409:Alanine / Q000378:metabolism* / Q000494:pharmacology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017124:Conserved Sequence; D006639:Histidine / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013211:Staphylococcus aureus / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000494:pharmacology
D001426:Bacterial Proteins; D050505:Mutant Proteins; D014538:Uridine Diphosphate N-Acetylmuramic Acid; D006639:Histidine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000409:Alanine