Studies on Deacetoxycephalosporin C Synthase Support a Consensus Mechanism for 2-Oxoglutarate Dependent Oxygenases

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• 作者：Hanna Tarhonskaya ; Andrea Sz枚ll枚ssi ; Ivanhoe K. H. Leung ; Jacob T. Bush ; Luc Henry ; Rasheduzzaman Chowdhury ; Aman Iqbal ; Timothy D. W. Claridge ; Christopher J. Schofield ; Emily Flashman
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：April 22, 2014
• 年：2014
• 卷：53
• 期：15
• 页码：2483-2493
• 全文大小：522K
• 年卷期：v.53,no.15(April 22, 2014)
• ISSN：1520-4995

文摘

Deacetoxycephalosporin C synthase (DAOCS) catalyzes the oxidative ring expansion of penicillin N (penN) to give deacetoxycephalosporin C (DAOC), which is the committed step in the biosynthesis of the clinically important cephalosporin antibiotics. DAOCS belongs to the family of non-heme iron(II) and 2-oxoglutarate (2OG) dependent oxygenases, which have substantially conserved active sites and are proposed to employ a consensus mechanism proceeding via formation of an enzyme路Fe(II)路2OG路substrate ternary complex. Previously reported kinetic and crystallographic studies led to the proposal of an unusual 鈥減ing-pong鈥?mechanism for DAOCS, which was significantly different from other members of the 2OG oxygenase superfamily. Here we report pre-steady-state kinetics and binding studies employing mass spectrometry and NMR on the DAOCS-catalyzed penN ring expansion that demonstrate the viability of ternary complex formation in DAOCS catalysis, arguing for the generality of the proposed consensus mechanism for 2OG oxygenases.