| Lipids are important for cellular function, both as constituents of membranes and as precursors of signaling molecules. The work described here has involved crystallographic studies of two enzymes which function in lipid metabolism. Besides revealing new information about the enzymes themselves, the two projects highlight the challenges of conducting crystallographic studies with integral membrane proteins (e.g. PGHS-1), as compared to soluble proteins (e.g. SQD1).; Prostaglandin H2 synthase-1 (PGHS-1), an integral membrane protein of the endoplasmic reticulum and nuclear envelope, catalyzes the committed step in formation of prostanoids. Its isoform, PGHS-2, performs the same reaction and is a key target of new pharmaceuticals designed to control inflammation, arthritis and possibly cancer. Although the crystal structure of PGHS-1 was solved in 1994, technical difficulties were slowing further progress. Therefore, the methods supporting structural biology (protein purification, crystallization and crystal mounting) were modified to reduce the expenditure of time and labor, while increasing the likelihood of success at each step. These improved techniques have begun to bear fruit in the form of more and better PGHS-1 crystal structures.; The sulfolipid sulfoquinovosyldiacylglycerol (SQDG) is nearly ubiquitous among photosynthetic organisms. SQDG production depends on a conserved enzyme, termed SQDB in bacteria and SQD1 in plants, which converts UDP-glucose and sulfite to UDP-sulfoquinovose. In this work, the crystal structure of SQD1 was determined. The overall protein fold confirmed its membership in the short-chain dehydrogenase/reductase (SDR) enzyme family. In the crystal structure, SQD1 binds both NAD+ and UDP-glucose in an unreacted, “poised” state. This pause in the catalytic cycle may be due to misorientation of the nicotinamide ring of NAD+, and presumably prevents untoward reactions from occurring before the sulfur donor has bound. Several amino acid residues were hypothesized to be important for the function of SQD1, particularly His183 and a “catalytic triad” of Thr145, Tyr182 and Lys186. All SQD1 structures have significant distortion of the Tyr182 phenol ring, which may support the theory that SDRs generally rely on an O η-deprotonated tyrosine to initiate catalysis.; Thr145 is hypothesized to accelerate catalysis forming by low-barrier hydrogen bonds (LBHBs) to UDP-glucose and/or reaction intermediates. A T145A mutant had no detectable activity in vitro, using UDP-glucose and inorganic sulfite as substrates. T145A protein, treated with UDP-glucose and sulfite, was crystallized. Unexpectedly, the product UDP-sulfoquinovose was observed in the active site, demonstrating that the mutant enzyme retains residual activity. A number of future experiments are suggested. |
