The Carboxyl Terminus of Eremomycin Facilitates Binding to the Non-d-Ala-d-Ala Segment of the Peptidoglycan Pentapeptide Stem

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• 作者：James Chang ; Hongyu Zhou ; Maria Preobrazhenskaya ; Peng Tao ; Sung Joon Kim
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：June 21, 2016
• 年：2016
• 卷：55
• 期：24
• 页码：3383-3391
• 全文大小：524K
• 年卷期：0
• ISSN：1520-4995

文摘

Glycopeptide antibiotics inhibit cell wall biosynthesis in Gram-positive bacteria by targeting the peptidoglycan (PG) pentapeptide stem structure (l-Ala-d-iso-Gln-l-Lys-d-Ala-d-Ala). Structures of the glycopeptide complexed with a PG stem mimic have shown that the d-Ala-d-Ala segment is the primary drug binding site; however, biochemical evidence suggests that the glycopeptide–PG interaction involves more than d-Ala-d-Ala binding. Interactions of the glycopeptide with the non-d-Ala-d-Ala segment of the PG stem were investigated using solid-state nuclear magnetic resonance (NMR). LCTA-1421, a double ¹⁵N-enriched eremomycin derivative with a C-terminal [¹⁵N]amide and [¹⁵N]Asn amide, was complexed with whole cells of Staphylococcus aureus grown in a defined medium containing l-[3-¹³C]Ala and d-[1-¹³C]Ala in the presence of alanine racemase inhibitor alaphosphin. ¹³C{¹⁵N} and ¹⁵N{¹³C} rotational-echo double-resonance (REDOR) NMR measurements determined the ¹³C–¹⁵N internuclear distances between the [¹⁵N]Asn amide of LCTA-1421 and the ¹³C atoms of the bound d-[1-¹³C]Ala-d-[1-¹³C]Ala to be 5.1 and 4.8 Å, respectively. These measurements also determined the distance from the C-terminal [¹⁵N]amide of LCTA-1421 to the l-[3-¹³C]Ala of PG to be 3.5 Å. The measured REDOR distance constraints position the C-terminus of the glycopeptide in the proximity of the l-Ala of the PG, suggesting that the C-terminus of the glycopeptide interacts near the l-Ala segment of the PG stem. In vivo REDOR measurements provided structural insight into how C-terminally modified glycopeptide antibiotics operate.