PMID- 10625675
OWN - NLM
STAT- MEDLINE
DCOM- 20000218
LR  - 20210209
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 275
IP  - 2
DP  - 2000 Jan 14
TI  - Identification of amino acid determinants of the positional specificity of mouse 
      8S-lipoxygenase and human 15S-lipoxygenase-2.
PG  - 1287-93
AB  - Phorbol ester-inducible mouse 8S-lipoxygenase (8-LOX) and its human homologue,
      15S-lipoxygenase-2 (15-LOX-2), share 78% identity in amino acid sequences, yet
      there is no overlap in their positional specificities. In this study, we
      investigated the determinants of positional specificity using a random
      chimeragenesis approach in combination with site-directed mutagenesis. Exchange
      of the C-terminal one-third of the 8-LOX with the corresponding portion of
      15-LOX-2 yielded a chimeric enzyme with exclusively 15S-lipoxygenase activity.
      The critical region was narrowed down to a cluster of five amino acids by
      expression of multiple cDNAs obtained by in situ chimeragenesis in Escherichia
      coli. Finally, a pair of amino acids, Tyr(603) and His(604), was identified as
      the positional determinant by site-directed mutagenesis. Mutation of both of
      these amino acids to the corresponding amino acids in 15-LOX-2 (Asp and Val,
      respectively) converted the positional specificity from 8S to 90% 15S without
      yielding any other by-products. Mutation of the corresponding residues in
      15-LOX-2 to the 8-LOX sequence changed specificity to 50% oxygenation at C-8 for 
      one amino acid substitution and 70% at C-8 for the double mutant. Based on the
      crystal structure of the reticulocyte 15-LOX, these two amino acids lie opposite 
      the open coordination position of the catalytic iron in a likely site for
      substrate binding. The change from 8 to 15 specificity entails a switch in the
      head to tail binding of substrate. Enzymes that react with substrate "head first"
      (5-LOX and 8-LOX) have a bulky aromatic amino acid and a histidine in these
      positions, whereas lipoxygenases that accept substrates "tail first" (12-LOX and 
      15-LOX) have an aliphatic residue with a glutamine or aspartate. Thus, this
      positional determinant of the 8-LOX and 15-LOX-2 may have significance for other 
      lipoxygenases.
FAU - Jisaka, M
AU  - Jisaka M
AD  - Division of Clinical Pharmacology, Vanderbilt University School of Medicine,
      Nashville, Tennessee 37232-6602, USA. jisaka@life.shimane-u.ac.jp
FAU - Kim, R B
AU  - Kim RB
FAU - Boeglin, W E
AU  - Boeglin WE
FAU - Brash, A R
AU  - Brash AR
LA  - eng
GR  - GM-53638/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (DNA Primers)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (Recombinant Proteins)
RN  - EC 1.13.11.- (Arachidonate Lipoxygenases)
RN  - EC 1.13.11.33 (Arachidonate 15-Lipoxygenase)
RN  - EC 1.13.11.40 (arachidonate 8-lipoxygenase)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Arachidonate 15-Lipoxygenase/*chemistry/genetics/*metabolism
MH  - Arachidonate Lipoxygenases/*chemistry/genetics/*metabolism
MH  - Base Sequence
MH  - Binding Sites
MH  - Crystallography, X-Ray
MH  - DNA Primers
MH  - Escherichia coli
MH  - Humans
MH  - Mammals
MH  - Mice
MH  - Models, Molecular
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - Protein Structure, Secondary
MH  - Recombinant Fusion Proteins/chemistry/metabolism
MH  - Recombinant Proteins/chemistry/metabolism
MH  - Restriction Mapping
MH  - Sequence Alignment
MH  - Sequence Homology, Amino Acid
MH  - Substrate Specificity
EDAT- 2000/01/08 09:00
MHDA- 2000/02/26 09:00
CRDT- 2000/01/08 09:00
PHST- 2000/01/08 09:00 [pubmed]
PHST- 2000/02/26 09:00 [medline]
PHST- 2000/01/08 09:00 [entrez]
AID - 10.1074/jbc.275.2.1287 [doi]
AID - S0021-9258(18)31247-X [pii]
PST - ppublish
SO  - J Biol Chem. 2000 Jan 14;275(2):1287-93. doi: 10.1074/jbc.275.2.1287.