A. 脂肪酶的C端之結構對於較長鏈受質的結合之重要性B. 雙磷酸鹽藥物對於四異戊二烯焦磷酸合成酶的抑制效果在結構上的分析

Part A. Lipase is a water-soluble enzyme that catalyzes the hydrolysis of ester bonds in water-insoluble lipid substrates. As most carboxylic ester hydrolases, Lipases contain a Ser-His-Asp catalytic triad similar to the much studied serine proteases. They also share a common structural framework alpha/beta hydrolase fold. In recent years lipases have attracted much interest because of their potential use in industrial applications. Several crystal structures of AFL, a novel lipase from the archaeon Archaeoglobus fulgidus, complexed with various ligands, have been determined at about 1.8 A resolution. This enzyme has optimal activity in the temperature range of 70-90 ℃ and pH 10-11. AFL consists of an N-terminal alpha/beta-hydrolase fold domain, a small lid domain, and a C-terminal beta-barrel domain. The N-terminal catalytic domain consists of a 6-stranded beta-sheet flanked by seven alpha-helices, four on one side and three on the other side. The C-terminal lipid binding domain consists of a beta-sheet of 14 strands and a substrate covering motif on top of the highly hydrophobic substrate binding site. The catalytic triad residues (Ser136, Asp163, and His210) and the residues forming the oxyanion hole (Leu31 and Met137) are in positions similar to those of other lipases. Long-chain lipid is located across the two domains in the AFL-substrate complex. Structural comparison of the catalytic domain of AFL with a homologous lipase from Bacillus subtilis reveals an opposite substrate binding orientation in the two enzymes. The presence of a large hydrophobic tunnel in the C-terminal domain in AFL enables it to have a higher preference toward long-chain substrates. The unusually large interacting surface area between the two domains may contribute to thermostability of the enzyme. Two amino acids, Asp 61 and Lys101, are identified as hinge residues regulating movement of the lid domain. The hydrogen-bonding pattern associated with these two residues is pH dependent, which may account for the optimal enzyme activity at high pH. We defined the role of the C-terminal β-barrel domain of the AFL as an anchoring domain for its substrate as well as providing the stability force. Further engineering of this novel lipase with high temperature and alkaline stability will find its use in industrial applications. Part B. Geranylgeranyl pyrophosphate synthase (GGPPS) catalyzes a condensation reaction of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to generate C20 geranylgeranyl pyrophosphate (GGPP), which is a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaeal ether linked lipids. We report several complexed structures of geranylgeranyl pyrophosphate synthase, a target for anticancer drugs. Bisphosphonate drugs used for osteoclast-mediated bone resorption and tumor-induced hypercalcemia are potent inhibitors of farnesyl pyrophosphate synthase (FPPS). Bisphosphonate drugs were shown to inhibit the activity of GGPPS. Bisphosphonates containing unbranched side chains bind to either the farnesyl diphosphate (FPP) substrate site or the geranylgeranyl diphosphate (GGPP) product site, and in one case, both sites, with the bisphosphonate moiety interacting with 3 Mg (2+) that occupy the same position as found in FPPS. However, each of three V-shaped bisphosphonates bind to both the FPP and GGPP sites. In each of the seven structures investigated, we found that there were up to four binding sites per monomer. These results show that some GGPPS inhibitors can occupy both substrate and product site and that binding modes as well as activity can be accurately predicted, facilitating the further development of GGPPS inhibitors as anticancer agents.