Studies on deacetoxycephalosporin C synthase support a consensus mechanism for 2-oxoglutarate dependent oxygenases.

Journal article

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·Fe(II)·2OG·substrate ternary complex. Previously reported kinetic and crystallographic studies led to the proposal of an unusual "ping-pong" 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.

Authors: Tarhonskaya, H; Szöllössi, A; Leung, I; Bush, J; Henry, L

• Publication status: Published
• Publisher: American Chemical Society
• Journal: Biochemistry
• Volume: 53
• Issue: 15
• Pages: 2483-2493
• Publication date: 2014-04-01
• DOI: 10.1021/bi500086p
• EISSN: 1520-4995
• ISSN: 0006-2960
• Language: English
• Keywords: Mass Spectrometry; Nuclear Magnetic Resonance, Biomolecular; Intramolecular Transferases; Ketoglutaric Acids; Crystallography, X-Ray; Penicillin-Binding Proteins; Catalysis; Kinetics; Oxygenases