THE CRYSTAL STRUCTURE OF Asn⁶⁹⁴His MUTANT OF L-1 ISOZYME OF SOYBEAN LIPOXYGENASE. K.Lewinski*, J.Steczko⁺, T.Holman⁽, E.Sigal⁽, B.Stec⁽, B.Axelrod⁺, W.Minor*. *University of Virginia, ⁺Purdue University, ⁽University of California-Riverside, ⁽Boston College, ⁽Mercator Genetics

Lipoxygenases are non-heme, non-sulfur iron dioxygenases catalyzing the hydroperoxidation of polyunsaturated fatty acids as linoleic, linolenic and arachidonic acid, the precursor of a number of physiologically potent effectors critical in human physiology. In soybeans there are three classes of lipoxygenase L-1, L-2 and L-3, all containing one iron atom per molecule and exhibiting >70% similarity in sequence.

Low temperature crystallographic studies at 1.4 Å resolution showed that in the L-1 isozyme the iron atom is coordinated octahedrally to three histidines (His⁴⁹⁹, His⁵⁰⁴ and His⁶⁹⁰), the C-terminal carboxylate group of Ile⁸³⁹, a water molecule and the O[[sigma]]₁ of Asn⁶⁹⁴. As the distance of Asn⁶⁹⁴ to iron was significantly larger than for histidine ligands (3.0 Å vs. 2.3 Å) the role of this residue in iron binding and mechanism of activity remained uncertain.

In some lipoxygenase sequences the position equivalent to Asn⁶⁹⁴ is occupied by a histidine residue. Recent studies of the L-3 isozyme in which asparagine was substituted by histidine showed no significant changes in activity. This result is contrary to our studies of Asn⁶⁹⁴His mutant of L-1 isozyme which shows only very limited activity.

The crystal structure of the Asn⁶⁹⁴His mutant is to high degree isomorphous to structure of wt-enzyme. Diffraction data were collected at 100K with the use of a standard RAXIS-II set-up. The position of His⁶⁹⁴ was found on the omit map in close vicinity of the iron atom. Refinement of the structure was performed in the resolution range 10-2 Å using the XPLOR program.

The iron atom is octahedrally coordinated by four histidine residues, including His⁶⁹⁴, the carboxyl end of Ile⁸³⁹ and a water molecule. Changes in geometry of the mutated enzyme and their impact on activity will be discussed.