The X‐Ray Structure and Biophysical Studies of a 15‐Lipoxygenase<sup>a</sup>

Boydigton, J. C.; Gaffney, Betty J.; Amzel, L. Mario; Doctor, Kutbuddin S.; Mavrophilipos, D.V.; Mavrophilipos, Zacharias V.; Colom, Adai; Yuan, Shao-Min

doi:10.1111/j.1749-6632.1994.tb52749.x

Cited by 4 publications

(4 citation statements)

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“…The crystal structure of LOX-1 shows that iron is buried within the enzyme fold and it is connected with the surface only by a narrow channel lined with hydrophobic amino acid residues (12,48). Therefore, enhanced catalytic activity of miniLOX can be reasonably explained on the basis of the highresolution structural data of the full-length LOX-1 (Protein Data Bank codes: 2sbl and 1f8n) (11,12).…”

Section: Discussionmentioning

confidence: 99%

A novel role for iron in modulating the activity and membrane‐binding ability of a trimmed soybean lipoxygenase‐1

et al. 2010

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Lipoxygenases (LOXs) are iron-containing enzymes that play critical roles in plants and animals. As yet, metal atom extraction, reconstitution, and substitution have not been successfully applied to soybean LOX-1 [Glycine max (L.) Merrill], a prototype member of the LOX family that is widely used in structural and kinetic studies. Here, tryptic digestion of native LOX-1, used as a control, allowed us to isolate the 60-kDa C-terminal region (termed miniLOX), that retains the catalytically active iron in a more accessible position. Then, iron was removed to obtain an unprecedented apo-miniLOX, which was reconstituted and substituted with different metal ions. These forms of miniLOX were characterized vs. native LOX-1 by kinetic analysis, near UV circular dichroism, steady-state fluorescence, and fluorescence resonance energy transfer. MiniLOX showed a 2-fold increase in the membrane-binding affinity compared with native LOX-1 and a remarkable 4-fold increase compared with apo-miniLOX (K(d)=9.2+/-1.0, 17.9+/-2.0, and 45.4+/-4.3 microM, respectively). Furthermore, miniLOX reconstituted with Fe(II) or Fe(III) partially recovered its membrane-binding ability (K(d)=21.4+/-2.4 and 18.9+/-5.5 microM, respectively), overall supporting a novel noncatalytic role for iron in the LOX family.

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Section: Discussionmentioning

confidence: 99%

A novel role for iron in modulating the activity and membrane‐binding ability of a trimmed soybean lipoxygenase‐1

et al. 2010

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“…The more tightly wrapped 3 10 -helix with i , i +3 hydrogen bonds (compared to i , i +4 in the α-helix) also occurs frequently, particularly at the ends of α-helices. On the other hand, the more loosely wrapped π-helix (with i , i +5 hydrogen bonds) is rare. − It has been suggested that π-helices only form when stabilized by very specific interactions (for example, side chain−metal ion interactions have been tailored to stabilize an isolated π-helix in solution , ). There have been several reports of π-helical conformations in molecular dynamics simulations of peptides. − However, Feig et al have recently suggested that these are the result of force field artifacts .…”

Section: Introductionmentioning

confidence: 99%

π-Helix Preference in Unsolvated Peptides

et al. 2004

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Ion mobility measurements have been used to examine helix formations in the gas phase for a series of alanine/glycine-based peptides that incorporate a glutamic acid (E) and lysine (K) at various positions along the backbone. Incorporation of an EK pair lowers the percent helix for all positions (presumably because hydrogen bonding between the backbone and the E and K side chains stabilize the nonhelical globular conformations). The largest percent helix is found when the EK pair is in an i,i+5 arrangement, which suggests that the preferred helical conformation for these peptides is a pi-helix. This conclusion is supported by comparison of cross sections deduced from the ion-mobility measurements to average cross sections calculated for conformations obtained from molecular dynamics simulations. The glutamic acid and lysine may form an ion pair that is stabilized by interactions with the helix macro-dipole.

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“…For approximately the first 30 years of protein structure determination, no π-helices were identified in the protein crystal structure literature. In the past 15 years, however, well-characterized π-helical turns have been identified in catalase (6), fumarase (7), P450 cytochrome (8), and soybean lipoxygenase (9,10). In soybean lipoxygenase, there are, in fact, two π-helices, one of which is continuous over 14 consecutive residues.…”

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confidence: 99%

“…Kaiser and Ke ´zdy proposed that amphiphilic π-helices might be stabilized by an interfacial environment (e.g., lipidwater or air-water interface) (16). The π-helix of soybean lipoxygenase appears to be stabilized by the binding of an Fe 2+ ion by histidine residues with this spacing (10). In this paper, we exploit both of these features and describe the design, synthesis, and characterization of a π-helix peptide.…”

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confidence: 99%

A Designed Zn²⁺-Binding Amphiphilic Polypeptide: Energetic Consequences of π-Helicity

Morgan¹,

Lynn²,

Miller-Auer³

et al. 2001

Biochemistry

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The pi-helix is a secondary structure with 4.4 amino acids per helical turn. Although it was proposed in 1952, no experimental support for its existence was obtained until the mid-1980s. While short peptides are unlikely to assume a marginally stable secondary structure spontaneously, they might do so in the presence of appropriate structural constraints. In this paper, we describe a peptide that is designed to assume a pi-helical conformation when stabilized by cetyltrimethylammonium bromide (CTAB) micelles and Zn(2+). In the designed peptide, lipophilic amino acids are placed such that it would be amphiphilic in the pi-helical, but not in the alpha-helical, conformation. Also, two His residues are incorporated with i, i + 5 spacing, designed to allow binding of Zn(2+) in a pi-helical but not an alpha-helical conformation. The peptide was found to form moderately stable monolayers at the air-water interface, with a collapse pressure that almost doubled when there was Zn(2+) in the subphase. Also, CTAB micelles induced a marked increase in the helicity of the peptide. In 50% TFE, the peptide had a CD spectrum consistent with an alpha-helical structure. The addition of 1 mM Zn(2+) to this solvent caused a saturable decline in ellipticity to approximately half of its original value. The peptide also bound Zn(2+) when it was bound to CTAB micelles, with Zn(2+) again inducing a decrease in ellipticity. The peptide had slightly greater affinity for Zn(2+) in the presence of the CTAB than in a 50% TFE solution (K(d) = 3.1 x 10(-4) M in CTAB and 2.3 x 10(-4) M in TFE). van't Hoff analysis indicated that thermal denaturation of the peptide in 50% TFE containing 1 mM Zn(2+) was associated with both enthalpic and entropic changes that were greater than those in the absence of Zn(2+). These observations are all consistent with the proposal that the peptide assumed a pi-helical conformation in the presence of Zn(2+) and CTAB micelles, and has allowed the stability of this rare conformation to be assessed.

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The X‐Ray Structure and Biophysical Studies of a 15‐Lipoxygenase^a

Cited by 4 publications

References 4 publications

A novel role for iron in modulating the activity and membrane‐binding ability of a trimmed soybean lipoxygenase‐1

A novel role for iron in modulating the activity and membrane‐binding ability of a trimmed soybean lipoxygenase‐1

π-Helix Preference in Unsolvated Peptides

A Designed Zn²⁺-Binding Amphiphilic Polypeptide: Energetic Consequences of π-Helicity

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The X‐Ray Structure and Biophysical Studies of a 15‐Lipoxygenasea

Cited by 4 publications

References 4 publications

A novel role for iron in modulating the activity and membrane‐binding ability of a trimmed soybean lipoxygenase‐1

A novel role for iron in modulating the activity and membrane‐binding ability of a trimmed soybean lipoxygenase‐1

π-Helix Preference in Unsolvated Peptides

A Designed Zn2+-Binding Amphiphilic Polypeptide: Energetic Consequences of π-Helicity

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The X‐Ray Structure and Biophysical Studies of a 15‐Lipoxygenase^a

A Designed Zn²⁺-Binding Amphiphilic Polypeptide: Energetic Consequences of π-Helicity