On the Relationships of Substrate Orientation, Hydrogen Abstraction, and Product Stereochemistry in Single and Double Dioxygenations by Soybean Lipoxygenase-1 and Its Ala542Gly Mutant

Abstract

Recent findings associate the control of stereochemistry in lipoxygenase (LOX) catalysis with a conserved active site alanine for S configuration hydroperoxide products, or a corresponding glycine for R stereoconfiguration. To further elucidate the mechanistic basis for this stereocontrol we compared the stereoselectivity of the initiating hydrogen abstraction in soybean LOX-1 and an Ala542Gly mutant that converts linoleic acid to both 13S and 9R configuration hydroperoxide products. Using 11R-3 Hand 11S-3 H-labeled linoleic acid substrates to examine the initial hydrogen abstraction, we found that all the primary hydroperoxide products were formed with an identical and highly stereoselective pro-S hydrogen abstraction from C-11 of the substrate (97-99% pro-S-selective). This strongly suggests that 9R and 13S oxygenations occur with the same binding orientation of substrate in the active site, and as the equivalent 9R and 13S products were formed from a bulky ester derivative (1-palmitoyl-2-linoleoylphosphatidylcholine), one can infer that the orientation is tail-first. Both the EPR spectrum and the reaction kinetics were altered by the R product-inducing Ala-Gly mutation, indicating a substantial influence of this Ala-Gly substitution extending to the environment of the active site iron. To examine also the reversed orientation of substrate binding, we studied oxygenation of the 15S-hydroperoxide of arachidonic acid by the Ala542Gly mutant soybean LOX-1. In addition to the usual 5S,15S-and 8S,15S-dihydroperoxides, a new product was formed and identified by high-performance liquid chromatography, UV, gas chromatography-mass spectrometry, and NMR as 9R,15S-dihydroperoxyeicosa-5Z,7E,11Z,13E-tetraenoic acid, the R configuration "partner" of the normal 5S,15S product. This provides evidence that both tail-first and carboxylate end-first binding of substrate can be associated with S or R partnerships in product formation in the same active site.

Dynamic Behavior of Fatty Acid Spin Labels within a Binding Site of Soybean Lipoxygenase-1

“…Perhaps the bulk of the 5-DSA doxyl ring, separated by only three carbons from the carboxylate, interferes with optimal interaction with a base, compared to the situation for 8-, 10-and 12-DSA. However, given the evident hydrophobic effect in binding thermodynamics of DSA molecules, and the fact that esterified fatty acids are good substrates (15,20,29), a specific charge interaction of the polar end of fatty acids with the protein may be relatively unimportant, or dynamic on the EPR time scale (μs to ns), for lipoxygenase substrate/product binding. It is possible that motion of the polar end of a fatty acid between multiple weak binding spots on the surface may assist in driving the hydrocarbon end of the substrate into the non-polar channel and in repositioning the peroxyl radical catalytic intermediate so that it can reoxidize iron.…”

“…A different category of model places the polar end of substrate near the protein surface, close to one suggested entrance to the cavity in SBL1 between Thr 259 and Leu541 (9). In this arrangement, the hydrophobic end of the substrate lies deeply in the binding pocket (18,20), as shown in Figure 1, essentially in the reverse orientation from that of product in the purple lipoxygenase structure (12). In fact, the stereochemistry of products from double dioxygenations of arachidonic acid demonstrates that substrates can position correctly for catalysis in two orientations: with the methyl end of the substrate chain entering the cavity most deeply or, alternatively, with the carboxyl end entering deepest (20).…”

“…Lipoxygenases from both plant and animal sources have overall structural similarity with a long internal cavity that passes by the catalytic iron ion, based on available X-ray structures (8)(9)(10)(11)(12)(13)(14). Some determinants of substrate binding within this cavity have been examined by mutation and modeling (15)(16)(17)(18)(19)(20), but there is little direct experimental evidence about how fatty acids enter or interact with the cavity. In this report, we take a spectroscopic approach, with spin labeled fatty acids, and examine the dynamic behavior of fatty acids bound to soybean lipoxygenase-1 (SBL1), the most studied representative of the family of LOX enzymes.…”

“…Referring to the helices of SBL1, this region begins between helices-2 and -11 and extends over the side of metal-binding helix-9 where water is coordinated to iron. Six to eight methylenes can be modeled in a hydrophobic cavity lying between iron and residues on helix-11, Ile538 or Leu541 (18,20). This hydrophobic region, between the surface and iron, is a likely site for binding an aliphatic chain.…”

Oxygen Activation in Lipoxygenase Model Reaction Studied with Density Functional Theory

“…DFT calculations also have revealed that the similar bond formation between the oxygen molecule and the carbon atom of 1,4-pentadiene moiety proceeds in the models of lipoxygenase (Figure 1) for both the Fe(II) (low-spin) and Fe(III) (high-spin) states when the 1,4-pentadiene moiety is deprotonated, and the optimized structure of the resulted ferric state-model containing hydroperoxy-derivative of 1,4-pentadiene is illustrated in Figure 3；this should correspond to a purple Fe III -OOL intermediate observed in SLO (Brash, 1999). As the hydroperoxy-derivative of unsaturated fatty acid has been used to obtain a ferric state lipoxygenase from a ferrous state in vitro (Goffa et al, 2005), it seems quite likely that the oxidation of the ferrous state to a ferric state proceeds through the reaction between the hydroperoxy-derivative formed at the first stage and Fe(II) ion in the native lipoxygenase, and the ferric state lipoxygenase thus formed should play an important role to produce the hydroperoxy-derivatives of fatty acid after the formation of iron(III) lipoxygenase. …”