Type of Document Thesis Author Imber, Ann Nicole Author's Email Address firstname.lastname@example.org URN etd-04072006-120440 Title Structure Variations in Lipoxygenase: Mutagenesis of Soybean Lipoxygenase-1 Degree Master of Science Department Biological Science, Department of Advisory Committee
Advisor Name Title Betty J. Gaffney Committee Member Hank W. Bass Committee Member Kenneth A. Taylor Committee Member Keywords
- Protein Expression
Date of Defense 2006-03-27 Availability unrestricted AbstractThe focus of this study is a prototypical member of the lipoxygenase protein family (acc.
PF00305), soybean lipoxygenase-1 (LOX-1). I am interested in two aspects of this enzyme: a
comparison of its structure and sequence with lipoxygenase enzymes and the overall shape of the
active site cavity with respect to catalysis. The first topic is primarily focused on an unusual
member of the lipoxygenase protein family that binds manganese instead of iron, manganese
lipoxygenase (MnLO). In order to perform mutational studies on structural regions of
importance in LOX-1, the sequence of LOX-1 will be compared to the sequence of MnLO. In
addition, the known structure of LOX-1 will be used as a platform to model features suggested
by MnLO. This is possible because of the considerable sequence identity within the conserved motifs of lipoxygenase family members. The results of this approach include implications for the metal selectivity of lipoxygenase enzymes and the structural significance of the highly conserved ð-helix found in the metal binding region. The second topic centers on the origin of the positional and stereochemical specificity of the lipoxygenase catalytic products. Members of the lipoxygenase family catalyze the insertion of molecular oxygen to their long-chain fatty acid
substrates in a highly specific manner. The structural rationale for this chemical specificity is implicated in the overall shape of the active site cavity. A stereodeterminant influencing the cavity shape was recently identified as a single residue conserved as a Gly in R- specific lipoxygenases and an Ala in S- specific enzymes. By performing mutagenesis on this residue in the LOX-1 enzyme and evaluating its EPR spectra, it is possible to monitor a catalytically relevant change in the lipoxygenase active site cavity that is propagated to the metal ion. Thus, this study proposes to examine key variations in the lipoxygenase amino acid sequence, especially in terms of enzyme catalysis and the details of the active site cavity and its metal binding site.
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