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

Abstract

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.

Catalytic Mechanisms of Fe(II)- and 2-Oxoglutarate-dependent Oxygenases

“…1E). A DAOCS⅐2OG⅐substrate ternary complex is proposed to react with O 2 to form a ferryl intermediate that initiates the reaction (32). One hydrogen atom is abstracted from the C2 ␤-methyl group of the substrate to produce a methylene radical, which undergoes rearrangement leading to ring expansion with the radical repositioned to the ternary carbon (C3), and then desaturation completes the reaction, perhaps by hydrogen atom transfer to the Fe(III)-OH (20,78).…”

“…Carbapenem synthase (CarC) is used to describe both desaturation and epimerization reactions (29,30). Ring expansion by deacetoxycephalosporin C synthase (DAOCS) (31,32), epoxidation by hyoscyamine 6␤-hydroxylase (H6H) (33), and endoperoxide formation by FtmOx1 acting on fumitremorgin B (34) are also briefly described.…”

O2 Activation by Non-Heme Iron Enzymes

“…27 Also it has been shown that DAOCS does, in fact, form a ternary complex in line with the general mechanistic strategy presented above. 48 …”

Reconstitution of TCA cycle with DAOCS to engineer Escherichia coli into an efficient whole cell catalyst of penicillin G

“…An in vitro process to carry out such a reaction would require the exogenous supply of 2OG that is a cost burden to the process; in addition the accumulation of the coproduct succinate could inhibit the bioconversion rate of the process (9). An in vivo process using E. coli as a whole-cell catalyst can provide an ideal solution to these problems because 2OG can be supplied in E. coli cells, and succinate will automatically be consumed without accumulation, thus effectively removing its inhibition of the DAOCS catalyzed reaction.…”

“…Several structures of DAOCS had been reported (7,8). Presteadystate kinetics and binding studies of DAOCS (9) suggested that DAOCS employs a mechanism similar to other 2OG-dependent oxygenases, where the binding of 2OG occurs first with subsequent formation of a ternary DAOCS·Fe(II)·2OG·penicillin substrate complex (9). The natural substrate of DAOCS is penicillin N, and its activity toward other penicillin analogs, such as penicillin G, is low (4).…”