Structural studies of a zinc-dependent deacetylase and a sesquiterpene cyclase

LpxC (UDP-{3-O-[(R)-3-hydroxymyristoyl]}- N-acetylglucosamine deacetylase) is a zinc-dependent deacetylase that hydrolyzes UDP-{3-O-[(R)-3-hydroxymyristoyl]}- N-acetylglucosamine to form acetate and UDP-{3-O-[(R)-3-hydroxymyristoyl]}glucosamine. LpxC catalyzes the first committed step in the biosynthesis of lipid A, a vital component of the outer membrane of Gram-negative bacteria. Accordingly, LpxC is an attractive target for the development of inhibitors that may serve as potential new antibiotics for the treatment of Gram-negative bacterial infections. The X-ray crystal structures of LpxC complexed with cacodylate (2.1 A resolution) or palmitate (2.7 A resolution) support the hypothesis that LpxC catalyzes deacetylation by using Glu78 and His265 as a general acid-base pair and the zinc-bound water as a nucleophile. The 2.7 A resolution structure of LpxC complexed with the substrate analogue inhibitor TU-514 allows for a detailed examination of the coordination of the catalytic zinc ion (Zn2+A) and other enzyme-inhibitor interactions in the active site. Evidence of a catalytic zinc ion in the native enzyme coordinated by H79, H238, D242, and two water molecules is provided by the 2.0 A resolution structure of LpxC complexed with imidazole. The 2.7 A resolution structure of LpxC complexed with UDP and the 3.1 A resolution structure of LpxC complexed with pyrophosphate provide the first view of interactions likely to be exploited by the substrate UDP group in the "basic patch" of the active site. Taken together, these structures provide a complete view of intermolecular interactions important for enzyme-substrate association in catalysis and for LpxC inhibitor design.;Gossypium arboreum (+)-delta-Cadinene Synthase. (+)-delta-Cadinene Synthase (DCS) catalyzes the cyclization of farnesyl diphosphate (FPP) to for the bicyclic sesquiterpene (+)-delta-cadinene. This is the first committed step in the biosynthesis of gossypol, an antimicrobial phytoalexin that protects cotton plants from bacterial and fungal invasion. The first three dimensional X-ray crystal structure of DCS was solved to 2.75 A resolution and reveals an alpha-helical terpene cyclase fold. DCS contains the so-called aspartate-rich motif DDXX(D,E) (boldface residues coordinate metal ions), but not the NSE/DTE metal-binding motif ((L,V)(V, L, A)-(N, D )D(L, I, V)X(S, T)XXXE) common to terpene cyclases. Instead, DCS contains a second aspartate-rich sequence, DDVAE, with the underlined residues highly conserved among the plant FPP cyclases (the boldface residues coordinate metal ions). The 2.75 A resolution structure of DCS complexed with the substrate analogue 2F-FPP reveals the binding of a tri-magnesium metal cluster and key enzyme-ligand intermolecular interactions in the active site. These structures provide the first evidence for a unique metal binding motif in a terpene cyclase and provide insight into possible substrate interactions and conformational changes that may occur in catalysis.