Biochemical and hydrogen-deuterium exchange studies of the single nucleotide polymorphism Y649C in human platelet 12-lipoxygenase linked to a bleeding disorder

Tran, Michelle; Signorelli, Rachel L.; Yamaguchi, Adriana; Chen, Eefie; Hollinstat, Michael; Iavarone, Anthony T.; Offenbacher, Adam R.; Holman, Theodore R.

doi:10.1016/j.abb.2022.109472

Cited by 4 publications

(3 citation statements)

References 70 publications

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“…A helical segment, consisting of residues 501–519, is located at least 24 Å from the site of mutation, with no direct backbone connection to helixes 419–441. However, HDX-detected allosteric networks, which are analogous in behavior to the altered long-range protein dynamics arising from catalytically altering site-selective mutations, often manifest at the “ends” of the dynamic pathway . The V426A-induced changes in the 501–510 peptide flexibility could be communicated via networks of side chain conformers in close contacts. , The fluctuations of both polar and nonpolar contacts can be responsible for heat transport in proteins, though displaying varying rates of heat transfer. , The sites of mutations and surrounding residues in the proximity of the active site are predominantly bulky aliphatic leucine and valine residues.…”

Section: Discussionmentioning

confidence: 99%

“…A helical segment, consisting of residues 501−519, is located at least 24 Å from the site of mutation, with no direct backbone connection to helixes 419−441. However, HDXdetected allosteric networks, which are analogous in behavior to the altered long-range protein dynamics arising from catalytically altering site-selective mutations, 61 often manifest at the "ends" of the dynamic pathway. 62 The V426A-induced changes in the 501−510 peptide flexibility could be communicated via networks of side chain conformers in close contacts.…”

Section: Mutation-dependent Tdhdx-ms Identifies Two Distinct Network ...mentioning

confidence: 99%

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Identification of the Thermal Activation Network in Human 15-Lipoxygenase-2: Divergence from Plant Orthologs and Its Relationship to Hydrogen Tunneling Activation Barriers

Ohler,

Taylor,

Bledsoe

et al. 2024

ACS Catal.

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The oxidation of polyunsaturated fatty acids by lipoxygenases (LOXs) is initiated by a C−H cleavage step in which the hydrogen atom is transferred quantum mechanically (i.e., via tunneling). In these reactions, protein thermal motions facilitate the conversion of ground-state enzyme−substrate complexes to tunneling-ready configurations and are thus important for transferring energy from the solvent to the active site for the activation of catalysis. In this report, we employed temperaturedependent hydrogen−deuterium exchange mass spectrometry (TDHDX-MS) to identify catalytically linked, thermally activated peptides in a representative animal LOX, human epithelial 15-LOX-2. TDHDX-MS of wild-type 15-LOX-2 was compared to two active site mutations that retain structural stability but have increased activation energies (E a ) of catalysis. The E a value of one variant, V427L, is implicated to arise from suboptimal substrate positioning by increased active-site side chain rotamer dynamics, as determined by X-ray crystallography and ensemble refinement. The resolved thermal network from the comparative E a s of TDHDX-MS between wild-type and V426A is localized along the front face of the 15-LOX-2 catalytic domain. The network contains a clustering of isoleucine, leucine, and valine side chains within the helical peptides. This thermal network of 15-LOX-2 is different in location, area, and backbone structure compared to a model plant lipoxygenase from soybean that exhibits a low E a value of catalysis compared to the human ortholog. The presented data provide insights into the divergence of thermally activated protein motions in plant and animal LOXs and their relationships to the enthalpic barriers for facilitating hydrogen tunneling.

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Section: Discussionmentioning

confidence: 99%

Section: Mutation-dependent Tdhdx-ms Identifies Two Distinct Network ...mentioning

confidence: 99%

Identification of the Thermal Activation Network in Human 15-Lipoxygenase-2: Divergence from Plant Orthologs and Its Relationship to Hydrogen Tunneling Activation Barriers

Ohler,

Taylor,

Bledsoe

et al. 2024

ACS Catal.

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“…Notably, the HDX behavior of the central region of helix α2, spanning residues 79–118, is virtually unchanged among the various glycan forms of Mo LOX (Figure S7), despite a change in substrate selectivity for the GLN variant. This observation is in stark contrast to the patterns of regional flexibility that are responsive to activity-altering mutations in plant and animal LOXs. , Specifically, the mutation to alanine of select large aliphatic residues lining the substrate entrance and binding channel in SLO increased off rates for substrate dissociation from the enzyme . HDX-MS analysis showed that increased mobility of helix α2, among a network of helices flanking the active site, may be related to the enhanced commitment to catalysis in SLO .…”

Section: Discussionmentioning

confidence: 99%

Impact of N-Glycosylation on Protein Structure and Dynamics Linked to Enzymatic C–H Activation in the M. oryzae Lipoxygenase

Whittington,

Sharma,

Hill

et al. 2024

Biochemistry

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Lipoxygenases (LOXs) from pathogenic fungi are potential therapeutic targets for defense against plant and select human diseases. In contrast to the canonical LOXs in plants and animals, fungal LOXs are unique in having appended N-linked glycans. Such important post-translational modifications (PTMs) endow proteins with altered structure, stability, and/or function. In this study, we present the structural and functional outcomes of removing or altering these surface carbohydrates on the LOX from the devastating rice blast fungus, M. oryzae, MoLOX. Alteration of the PTMs did notinfluence the active site enzyme−substrate ground state structures as visualized by electron−nuclear double resonance (ENDOR) spectroscopy. However, removal of the eight N-linked glycans by asparagine-toglutamine mutagenesis nonetheless led to a change in substrate selectivity and an elevated activation energy for the reaction with substrate linoleic acid, as determined by kinetic measurements. Comparative hydrogen−deuterium exchange mass spectrometry (HDX-MS) analysis of wild-type and Asn-to-Gln MoLOX variants revealed a regionally defined impact on the dynamics of the arched helix that covers the active site. Guided by these HDX results, a single glycan sequon knockout was generated at position 72, and its comparative substrate selectivity from kinetics nearly matched that of the Asn-to-Gln variant. The cumulative data from model glyco-enzyme MoLOX showcase how the presence, alteration, or removal of even a single N-linked glycan can influence the structural integrity and dynamics of the protein that are linked to an enzyme's catalytic proficiency, while indicating that extensive glycosylation protects the enzyme during pathogenesis by protecting it from protease degradation.

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Lipoxygenase Dynamics and Catalysis Studied by Temperature‐Dependent Hydrogen–Deuterium Exchange

Offenbacher,

Guy

2024

Encyclopedia of Inorganic and Bioinorganic Chemistry

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An active pursuit in biochemistry is the understanding of how the structure and dynamics of protein systems regulate their biological function. In this chapter, we describe temperature‐dependent hydrogen–deuterium exchange mass‐spectrometry (TDHDX‐MS) as a tool used to investigate the structure and dynamics of metalloenzymes in solution. While the method is applicable to many metalloproteins, this review will present TDHDX‐MS studies centered on the widely represented family of mononuclear, non‐heme iron containing lipoxygenase (LOX) enzymes that oxidize fatty acids to initiate biological reactions and activate cellular signaling. We will present how TDHDX has been used to describe the anisotropic nature of energy transfer in LOXs to initiate the rate‐limiting hydrogen atom transfer reaction that proceeds through a tunneling process. Further, TDHDX‐MS has been used detect protein motions and conformations in LOXs related to molecular recognition, protein allostery, and polymorphisms linked to disease.

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Biochemical and hydrogen-deuterium exchange studies of the single nucleotide polymorphism Y649C in human platelet 12-lipoxygenase linked to a bleeding disorder

Cited by 4 publications

References 70 publications

Identification of the Thermal Activation Network in Human 15-Lipoxygenase-2: Divergence from Plant Orthologs and Its Relationship to Hydrogen Tunneling Activation Barriers

Identification of the Thermal Activation Network in Human 15-Lipoxygenase-2: Divergence from Plant Orthologs and Its Relationship to Hydrogen Tunneling Activation Barriers

Impact of N-Glycosylation on Protein Structure and Dynamics Linked to Enzymatic C–H Activation in the M. oryzae Lipoxygenase

Lipoxygenase Dynamics and Catalysis Studied by Temperature‐Dependent Hydrogen–Deuterium Exchange

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