Conserved histidine residues in soybean lipoxygenase: functional consequences of their replacement.
Sequences of 13 lipoxygenases from various plant and mammalian species, thus far reported, display a motif of 38 amino acid residues which includes 5 conserved histidines and a 6th histidine about 160 residues downstream. These residues occur at positions 494, 499, 504, 522, 531, and 690 in soybean lipoxygenase isozyme L-1. Since the participation of iron in the lipoxygenase reaction has been established and existing evidence based on Mössbauer and EXAFS spectroscopy suggests that histidines may be involved in iron binding, the effect of the above residues has been examined in soybean lipoxygenase L-1. Six singly mutated lipoxygenases have been produced in which each of the His residues has been replaced with glutamine. Two additional mutants have been constructed wherein the codons for His-494 and His-504 have been replaced by serine codons. All of the mutant lipoxygenases, which were obtained by expression in Escherichia coli, have mobilities identical to that of the wild-type enzyme on denaturing gel electrophoresis and respond to lipoxygenase antibodies. The mutated proteins H499Q, H504Q, H504S, and H690Q are virtually inactive, while H522Q has about 1% of the wild-type activity. H494Q, H494S, and H531Q are about 37%, 8%, and 20% as active as the wild type, respectively. His-517 is conserved in the several lipoxygenase isozymes but not in the animal isozymes. The mutant H517Q has about 33% of the wild-type activity. The inactive mutants, H499Q, H504Q, H504S, and H690Q, become insoluble when heated for 3 min at 65 degrees C, as does H522Q. The other mutants and the wild-type are stable under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
Steczko
J
J
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
Donoho
G P
GP
Clemens
J C
JC
Dixon
J E
JE
Axelrod
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D007501:Iron; D008084:Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology*; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D006639:Histidine; D007501:Iron; D008084:Lipoxygenase