A novel assay of bacterial peptidoglycan synthesis for natural product screening.
Although a large number of microbial metabolites have been discovered as inhibitors of bacterial peptidoglycan biosynthesis, only a few inhibitors were reported for the pathway of UDP-MurNAc-pentapeptide formation, partly because of the lack of assays appropriate for natural product screening. Among the pathway enzymes, D-Ala racemase (Alr), D-Ala:D-Ala ligase (Ddl) and UDP-MurNAc-tripeptide:D-Ala-D-Ala transferase (MurF) are particularly attractive as antibacterial targets, because these enzymes are essential for growth and utilize low-molecular-weight substrates. Using dansylated UDP-MurNAc-tripeptide and L-Ala as the substrates, we established a cell-free assay to measure the sequential reactions of Alr, Ddl and MurF coupled with translocase I. This assay is sensitive and robust enough to screen mixtures of microbial metabolites, and enables us to distinguish the inhibitors for D-Ala-D-Ala formation, MurF and translocase I. D-cycloserine, the D-Ala-D-Ala pathway inhibitor, was successfully detected by this assay (IC(50)=1.7 microg ml(-1)). In a large-scale screening of natural products, we have identified inhibitors for D-Ala-D-Ala synthesis pathway, MurF and translocase I.
Murakami
Ryo
R
Exploratory Research Laboratories II, Daiichi-Sankyo Co., Ltd, Kitakasai, Edogawa-ku, Tokyo, Japan. murakami.ryo.bw@daiichisankyo.co.jp
Muramatsu
Yasunori
Y
Minami
Emiko
E
Masuda
Kayoko
K
Sakaida
Yoshiharu
Y
Endo
Shuichi
S
Suzuki
Takashi
T
Ishida
Osamu
O
Takatsu
Toshio
T
Miyakoshi
Shunichi
S
Inukai
Masatoshi
M
Isono
Fujio
F
D001419:Bacteria / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D001688:Biological Products / Q000737:chemistry / Q000494:pharmacology*; D002240:Carbohydrate Sequence; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D003523:Cycloserine / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D005285:Fermentation; D005453:Fluorescence; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010457:Peptidoglycan / Q000096:biosynthesis* / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives / Q000378:metabolism
D001688:Biological Products; D010457:Peptidoglycan; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C002225:UDP-N-acetylmuramic acid pentapeptide; D003523:Cycloserine; D010453:Peptide Synthases; C060628:UDP-N-acetylmuramoylalanyl-D-glutamyllysine-D-alanyl-D-alanine ligase; C019638:D-alanylalanine synthetase