Christa Gerth

Structural Basis for Lipoxygenase Specificity CONVERSION OF THE HUMAN LEUKOCYTE 5-LIPOXYGENASE TO A 15-LIPOXYGENATING ENZYME SPECIES BY SITE-DIRECTED MUTAGENESIS

Mammalian lipoxygenases constitute a heterogeneous family of lipid-peroxidizing enzymes, and the various isoforms are categorized with respect to their positional specificity of arachidonic acid oxygenation into 5-, 8-, 12-, and 15-lipoxygenases. Structural modeling suggested that the substrate binding pocket of the human 5-lipoxygenase is 20% bigger than that of the reticulocyte-type 15-lipoxygenase; thus, reduction of the active-site volume was suggested to convert a 5-lipoxygenase to a 15-lipoxygenating enzyme species. To test this “space-based” hypothesis of the positional specificity, the volume of the 5-lipoxygenase substrate binding pocket was reduced by introducing space-filling amino acids at critical positions, which have previously been identified as sequence determinants for the positional specificity of other lipoxygenase isoforms. We found that single point mutants of the recombinant human 5-lipoxygenase exhibited a similar specificity as the wild-type enzyme but double, triple, and quadruple mutations led to a gradual alteration of the positional specificity from 5S- via 8S- toward 15S-lipoxygenation. The quadruple mutant F359W/A424I/N425M/A603I exhibited a major 15S-lipoxygenase activity (85–95%), with (8S,5Z,9E,11Z,14Z)-8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid being a minor side product. These data indicate the principle possibility of interconverting 5- and 15-lipoxygenases by site-directed mutagenesis and appear to support the space-based hypothesis of positional specificity.

Expression of 12/15-Lipoxygenase Attenuates Intracellular Lipid Deposition During In Vitro Foam Cell Formation

Objectives—Lipoxygenases with different positional specificity have been implicated in atherogenesis, but the precise roles of the various isoforms remain unclear. Because of its capability of oxidizing low-density lipoprotein (LDL) to an atherogenic form, 12/15-lipoxygenases have been suggested to initiate LDL oxidation in vivo; thus, these enzymes may exhibit pro-atherogenic activities. However, in several rabbit atherosclerosis models, the enzyme appears to act atheroprotective. Methods and Results—To test the impact of 12/15-lipoxygenase expression on early atherogenesis, we established an in vitro foam cell model, which is based on the uptake of acetylated LDL by murine macrophages. In this system, we found that 12/15-lipoxygenase expression protects the cells from intracellular lipid deposition. This effect was related to an attenuated uptake of modified LDL, as indicated by impaired expression of scavenger receptor A and to accelerated intracellular lipid metabolism. Conclusions—Our results indicate that the role of 12/15-lipoxygenase in atherogenesis may not be restricted to oxidative LDL modification. Expression of this lipid-peroxidizing enzyme may impact both lipid uptake and intracellular lipid turnover. These data provide a plausible explanation for the antiatherogenic effect of 12/15-LOX in rabbit atherosclerosis models.

Nucleotide status in erythrocytes of rats infected with Plasmodium berghei.

Red blood cells (RBC) as well as plasmodia are not able to synthesize purines; therefore salvage pathways are essentiell for both. The source of purines is plasma adenosine. It enters the plasma membrane of erythrocytes via a specific transport protein which is located in band 4.5 together with the glucose transporter1. The parasites provide a high influx of adenosine into their infected host by altering the expression of the nucleoside transporter. In the erythrocyte adenosine is converted to AMP and to hypoxanthine. Last compound is preferentially translocated into parasites. They are characterized by a high capacity of purine nucleotide metabolizing enzymes including those of the salvage pathways. Parasites use hypoxanthine as substrate for the synthesis of all purine nucleotides. The enzymes of these reactions are associated with the plasmodial membrane. Therefore the question arises which interactions do exist in the purine nucleotide metabolism between the two compartments parasite and red blood cell. Is was our aim to clarify it by an analysis of the purine nucleotide status.