Key role of conserved histidines in recombinant mouse beta-carotene 15,15'-monooxygenase-1 activity.
Alignment of sequences of vertebrate beta-carotene 15,15'-monooxygenase-1 (BCMO1) and related oxygenases revealed four perfectly conserved histidines and five acidic residues (His172, His237, His308, His514, Asp52, Glu140, Glu314, Glu405, and Glu457 in mouse BCMO1). Because BCMO1 activity is iron-dependent, we propose that these residues participate in iron coordination and therefore are essential for catalytic activity. To test this hypothesis, we produced mutant forms of mouse BCMO1 by replacing the conserved histidines and acidic residues as well as four histidines and one glutamate non-conserved in the overall family with alanines by site-directed mutagenesis. Our in vitro and in vivo data showed that mutation of any of the four conserved histidines and Glu405 caused total loss of activity. However, mutations of non-conserved histidines or any of the other conserved acidic residues produced impaired although enzymatically active proteins, with a decrease in activity mostly due to changes in V(max). The iron bound to protein was determined by inductively coupled plasma atomic emission spectrometry. Bound iron was much lower in preparations of inactive mutants than in the wild-type protein. Therefore, the conserved histidines and Glu405 are absolutely required for the catalytic mechanism of BCMO1. Because the mutant proteins are impaired in iron binding, these residues are concluded to coordinate iron required for catalytic activity. These data are discussed in the context of the predicted structure for the related eubacterial apocarotenal oxygenase.
Poliakov
Eugenia
E
Laboratory of Retinal Cell and Molecular Biology, NEI, National Institutes of Health, Bethesda, MD 20892-0706, USA.
Gentleman
Susan
S
Cunningham
Francis X
FX
Miller-Ihli
Nancy J
NJ
Redmond
T Michael
TM
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013054:Spectrophotometry, Atomic; D014801:Vitamin A / Q000378:metabolism; D019207:beta Carotene / Q000378:metabolism; D050559:beta-Carotene 15,15'-Monooxygenase
D011994:Recombinant Proteins; D019207:beta Carotene; D014801:Vitamin A; D018698:Glutamic Acid; D006639:Histidine; D007501:Iron; D010105:Oxygenases; C493359:Bco1 protein, mouse; C493358:BCO1 protein, human; D050559:beta-Carotene 15,15'-Monooxygenase; D000409:Alanine