Structural origins of the selectivity of the trifunctional oxygenase clavaminic acid synthase

Clavaminate synthase (CAS), a remarkable Fe(II)/2-oxoglutarate oxygenase, c atalyzes three separate oxidative reactions in the biosynthesis of clavulan ic acid, a clinically used inhibitor of serine beta-lactamases, The first C AS-catalyzed step (hydroxylation) is separated from the latter two (oxidati ve cyclization/desaturation) by the action of an amidinohydrolase, Here, we describe crystal structures of CAS in complex with Fe(II), 2-oxoglutarate (2OG) and substrates (N-alpha-acetyl-L-arginine and proclavaminic acid). Th ey reveal how GAS catalyzes formation of the clavam nucleus, via a process unprecedented in synthetic organic chemistry, and suggest how it discrimina tes between substrates and controls reaction of its highly reactive ferryl intermediate. The presence of an unpredicted jelly roll beta-barrel core in CAS implies divergent evolution within the family of 2OG and related oxyge nases, Comparison with other non-heme oxidases/oxygenases reveals flexibili ty in the position which dioxygen ligates to the iron, in contrast to the a nalogous heme-using enzymes.