Structural and Functional Characterization of Second-Coordination Sphere Mutants of Soybean Lipoxygenase-1

Tomchick, Diana R.; Phan, Phuc; Cymborowski, M.; Minor, Władek; Holman, Theodore R.

doi:10.1021/bi002893d

Cited by 120 publications

(160 citation statements)

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“…In contrast to the other mutants in this study, Q495E and the wt enzyme exhibit an H-bond between His499 and position 495 (Figure 1). According to the crystal structures, His499 is mobile in the wt structure (14). It was speculated that the interaction between His499 and Glu495, in Q495E, positions Glu495 in a similar orientation as Gln495 in the wt structure (14).…”

Section: Resultsmentioning

confidence: 99%

“…According to the crystal structures, His499 is mobile in the wt structure (14). It was speculated that the interaction between His499 and Glu495, in Q495E, positions Glu495 in a similar orientation as Gln495 in the wt structure (14). The increased electrostatic repulsion in the active site may be the cause for the altered ligand field in the mutant as compared to wt sLO-1, at least in the resting state ( Figure 6).…”

Section: Resultsmentioning

confidence: 99%

“…A similar network is also present in 15-rLO (27), and it was speculated that interactions between the substrate and this hydrogen bond network are responsible for the observed sensitivity of the coordination number in ferrous sLOs (14). Previously, Gln697 and Gln495 were mutated (Q697E, Q697N, Q495E, and Q495A) ( Figure 1), and their kinetic properties were related to their crystal structures (14). This mutant data provided evidence that structural changes of the hydrogen bond network could account for the observed kinetic differences.…”

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confidence: 96%

“…Crystal structures for ferrous LOs have been solved for two soybean isoforms (sLO-1 and sLO-3) (14,(24)(25)(26) and rabbit reticulocyte 15-LO (15-rLO) (27). The three structures display a similar overall folding pattern.…”

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confidence: 99%

“…An examination of the crystal structure of wildtype (wt) sLO-1 (14) indicates the presence of an extensive hydrogen bonding network that connects the weakly bound asparagine ligand (Asn694) via the two second coordination sphere residues, Gln697 and Gln495, to an equatorial iron ligand, His499 (Figure 1). A similar network is also present in 15-rLO (27), and it was speculated that interactions between the substrate and this hydrogen bond network are responsible for the observed sensitivity of the coordination number in ferrous sLOs (14). Previously, Gln697 and Gln495 were mutated (Q697E, Q697N, Q495E, and Q495A) ( Figure 1), and their kinetic properties were related to their crystal structures (14).…”

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Spectroscopic Characterization of Soybean Lipoxygenase-1 Mutants: the Role of Second Coordination Sphere Residues in the Regulation of Enzyme Activity

et al. 2003

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Lipoxygenases are non-heme iron enzymes, which catalyze the stereo-and regiospecific hydroperoxidation of unsaturated fatty acids. Spectroscopic studies on soybean lipoxygenase have shown that the ferrous form of the enzyme is a mixture of five-and six-coordinate species (40 and 60%, respectively). Addition of substrate leads to a purely six-coordinate form. A series of mutations in the second coordination sphere (Q697E, Q697N, Q495A, and Q495E) were generated, and the structures of the mutants were solved by crystallography [Tomchick et al. (2001) Biochemistry 40, 7509-7517]. While this study clearly showed the contribution of H-bond interactions between the first and the second coordination spheres in catalysis, no correlation with the coordination environment of the Fe II was observed. A recent study using density-functional theory [Lehnert and Solomon (2002) J. Biol. Inorg. Chem. 8, 294-305] indicated that coordination flexibility, involving the Asn694 ligand, is regulated via H-bond interactions. In this paper, we investigate the solution structures of the second coordination sphere mutants using CD and MCD spectroscopy since these techniques are more sensitive indicators of the first coordination sphere ligation of Fe II systems. Our data demonstrate that the iron coordination environment directly relates to activity, with the mutations that have the ability to form a five-coordinate/six-coordinate mixture being more active. We propose that the H-bond between the weak Asn694 ligand and the Gln697 plays a key role in the modulation of the coordination flexibility of Asn694, and thus, is crucial for the regulation of enzyme reactivity.Lipoxygenases (LOs) 1 are mononuclear non-heme iron dioxygenases that catalyze the stereo-and regiospecific hydroperoxidations of cis,cis-1,4 polyunsaturated pentadienyl containing fatty acids. LOs have been isolated and characterized from numerous mammalian and plant sources and are believed to occur ubiquitously throughout these two phyla (1-3). In mammalian organisms, there are multiple LOs with differing regio-and stereospecificity against their substrate, arachidonic acid (AA). The products of these reactions are further metabolized by an array of enzymes leading to the formation of two major classes of cell effector molecules, leukotrienes and lipoxins, which are mediators in anaphylactic and inflammatory disorders (4, 5). Additionally, LO products are involved in a variety of metabolic functions, including organelle degradation (6), transcription regulation (7), and possibly tumor cell metastasis (8). For plant LOs, the main substrates are linoleic acid (LA) and linolenic acid, which are further metabolized to plant hormones, such as jasmonic acid. These hormones are involved in a variety of metabolic functions including plant growth and development, senescence, and cellular response to wounding and infections by pathogens (1). Soybeans have three LO isoforms (sLO-1, sLO-2, and sLO-3), which have homologous sequences and molecular weights of ∼96 kDa (9). This...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 96%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Spectroscopic Characterization of Soybean Lipoxygenase-1 Mutants: the Role of Second Coordination Sphere Residues in the Regulation of Enzyme Activity

et al. 2003

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Iron Proteins with Mononuclear Active Sites

Emerson¹,

Mehn²,

Que³

2005

Encyclopedia of Inorganic Chemistry

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Metalloenzymes containing a mononuclear nonheme iron center are often involved in dioxygen metabolism. We have divided this superfamily of enzymes into seven subcategories: iron superoxide dismutase and superoxide reductase, lipoxygenase, catechol dioxygenases, α‐keto acid‐dependent enzymes, isopenicillin N synthase, pterin‐dependent hydroxylases, and Rieske dioxygenases. The degree of catalytic versatility and coordination flexibility encompassed by these enzymes is unsurpassed by any other known metalloenzyme superfamily. Herein we discuss the present state of knowledge regarding their active site structures and the critical role the metal center plays in their respective catalytic mechanisms.

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