Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis

Abstract

Multiple lipoxygenase sequence alignments and structural modeling of the enzyme͞substrate interaction of the cucumber lipid body lipoxygenase suggested histidine 608 as the primary determinant of positional specificity. Replacement of this amino acid by a less-space-filling valine altered the positional specificity of this linoleate 13-lipoxygenase in favor of 9-lipoxygenation. These alterations may be explained by the fact that H608V mutation may demask the positively charged guanidino group of R758, which, in turn, may force an inverse head-to-tail orientation of the fatty acid substrate. The R758L؉H608V double mutant exhibited a strongly reduced reaction rate and a random positional specificity. Trilinolein, which lacks free carboxylic groups, was oxygenated to the corresponding (13S)-hydro(pero)xy derivatives by both the wild-type enzyme and the linoleate 9-lipoxygenating H608V mutant. These data indicate the complete conversion of a linoleate 13-lipoxygenase to a 9-lipoxygenating species by a single point mutation. It is hypothesized that H608V exchange may alter the orientation of the substrate at the active site and͞or its steric configuration in such a way that a stereospecific dioxygen insertion at C-9 may exclusively take place.Lipoxgenases (LOXs; linoleate:oxygen oxidoreductase; EC 1.13.11.12) are widely distributed in the plant and animal kingdom (1, 2). They constitute a family of nonheme ironcontaining dioxygenases that catalyze the regio-and stereoselective dioxygenation of polyenoic fatty acids forming hydroperoxy derivatives (3). In mammals, LOXs are classified according to their positional specificity of arachidonic acid oxygenation (2, 4). Because arachidonic acid either is not present in higher plants or is a minor constituent of cellular lipids, plant LOXs are classified into 9-and 13-LOXs with respect to their positional specificity of linoleic acid (LA) oxygenation (5). Recently, a more comprehensive classification of plant LOXs has been proposed based on the comparison of their primary structures (6).The positional specificity of LOXs is a result of two catalytic processes. (i) Regio-and stereospecific hydrogen removal; with substrate fatty acids containing several doubly allylic methylenes such as linolenic acid, arachidonic acid, or eicosapentaenoic acid hydrogen abstraction from two, three, or four doubly allylic methylenes, respectively, is possible. (ii) Regio-and stereospecific oxygen insertion: when hydrogen is abstracted from a certain doubly allylic methylene, molecular oxygen can be introduced either at the [ϩ2] or at the [Ϫ2] position (Fig. 1). Thus, a fatty acid containing three doubly allylic methylenes such as arachidonic acid can be oxygenated by a LOX to six regioisomeric hydroperoxy derivatives (HPETEs), namely 15-and 11-HPETE (originating from C-13 hydrogen removal), 12-and 8-HPETE (C-10 hydrogen removal), and 9-and 5-HPETE (C-7 hydrogen removal). Experiments on mammalian 12-and 15-LOXs indicated that the site of hydrogen abstraction can be altered when critical ...

On the Substrate Binding of Linoleate 9‐Lipoxygenases

“…According to the existing models, the residue in question is in close proximity to an arginine residue positioned on the bottom of the active site pocket. In 13-LOX the phenylalanine is thought to shield the positive charge of this arginine and lead to a preferential entry of the fatty acid with the methyl group first, leading to oxygenation at position C13 [5]. However, our data on StLOX1 suggest that the valine to phenylalanine exchange may not be sufficient to change the product specificity of C18 fatty acid conversion for this enzyme and therefore does not alter the favored substrate orientation in the active site of the enzyme.…”

“…Additionally, we tested the activity of StLOX1 against a number of esterified fatty acids as substrates. Trilinolein and triarachidonin were used as substrates under the conditions described for the cucumber lipid body LOX, CslbLOX [5], while 1,2-diarachidonoyl-sn-glycero-3-phosphatidylcholine, as has been described for other LOX enzymes [8,9]. We could not observe activity against any of the substrates, which suggests that binding of the substrate takes place with the carboxyl-group first.…”

“…In 9-LOX the amino acid aligning with this position is usually a less space-filling valine residue. It is is suggested that the presence of the smaller amino acid demasks a positively charged arginine residue, the so-called ''Hornung determinant'', at the bottom of the substrate pocket, which can stabilize an insertion of the substrate with the carboxylic group first in the active site pocket leading to a preferable oxygen insertion in position 9 [5].…”

Molecular analysis of lipoxygenase (LOX) genes in common wheat and phylogenetic investigation of LOX proteins from model and crop plants

“…Plant LOXs have been divided into two types, 9-LOXs and 13-LOXs, which catalyze oxygenation at the carbon atoms 9 and 13 of the hydrocarbon backbone of LA, respectively. However, this classification scheme has encountered difficulty because more and more animal and plant LOXs are found to yield a mixture of products upon catalysis (Brash, 1999;Hornung et al, 1999Hornung et al, , 2008. Phylogenetic analysis suggests that the LOX superfamily contains at least two distinct families, which are composed of the LOXs from animals and plants, respectively (Brash, 1999).…”

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

“…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.…”