Conversion of human 15-lipoxygenase to an efficient 12-lipoxygenase: the side-chain geometry of amino acids 417 and 418 determine positional specificity

Protein Eng. 1995 Mar;8(3):275-82. doi: 10.1093/protein/8.3.275.


Positional specificity determinants of human 15-lipoxygenase were examined by site-directed mutagenesis and by kinetic analysis of the wild-type and variant enzymes. By comparing conserved differences among sequences of 12- and 15-lipoxygenases, a small region responsible for functional differences between 12- and 15-lipoxygenases has been identified. Furthermore, the replacement of only two amino acids in 15-lipoxygenase (at 417 and 418 in the primary sequence) by those found in certain 12-lipoxygenases results in an enzyme that has activity similar to 12-lipoxygenase. An examination of the activity of nine variants of lipoxygenase demonstrated that the amino acid side-chain bulk and geometry of residues 417 and 418 are the key components of the positional specificity determinant of 15-lipoxygenase. Overexpression of a variant (containing valines at positions 417 and 418) that performs predominantly 12-lipoxygenation was achieved in a baculo-virus-insect cell culture system. This variant was purified to > 90% homogeneity and its kinetics were compared with the wild-type 15-lipoxygenase. The variant enzyme has no change in its apparent KM for arachidonic acid and a minor (3-fold) change in its Vmax. For linoleic acid, the variant has no change in its KM and a 10-fold reduction in its Vmax, as expected for an enzyme performing predominantly 12-lipoxygenation. The results are consistent with a model in which two amino acids of 15-lipoxygenase (isoleucine 417 and methionine 418) constitute a structural element which contributes to the regiospecificity of the enzyme. Replacement of these amino acids with those found in certain 12-lipoxygenases results in an enzyme which can bind arachidonic acid in a catalytic register that prefers 12-lipoxygenation.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Arachidonate 12-Lipoxygenase / chemistry
  • Arachidonate 12-Lipoxygenase / genetics
  • Arachidonate 12-Lipoxygenase / isolation & purification
  • Arachidonate 12-Lipoxygenase / metabolism*
  • Arachidonate 15-Lipoxygenase / chemistry
  • Arachidonate 15-Lipoxygenase / genetics*
  • Arachidonate 15-Lipoxygenase / isolation & purification
  • Arachidonate 15-Lipoxygenase / metabolism*
  • Arachidonic Acid / metabolism
  • Baculoviridae / genetics
  • Base Sequence
  • Chromatography, High Pressure Liquid
  • Escherichia coli / genetics
  • Humans
  • Kinetics
  • Linoleic Acid
  • Linoleic Acids / metabolism
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Recombinant Proteins / metabolism
  • Structure-Activity Relationship
  • Substrate Specificity


  • Linoleic Acids
  • Recombinant Proteins
  • Arachidonic Acid
  • Linoleic Acid
  • Arachidonate 12-Lipoxygenase
  • Arachidonate 15-Lipoxygenase