Key Role of Conserved Histidines in Recombinant Mouse β-Carotene 15,15′-Monooxygenase-1 Activity

Eugenia Poliakov,Susan Gentleman,Francis X Cunningham,Nancy J Miller-Ihli,T Michael Redmond

doi:10.1074/jbc.m500409200

Abstract

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.

Highlights

Alignment of sequences of vertebrate ␤-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)
BCMO1 belongs to a family of oxygenases of diverse activities, including lignostilbene dioxygenase in bacteria (8 –10); an epoxycarotenoid-cleaving enzyme required for abscisic acid biosynthesis in plants [11]; and mammalian RPE65, a retinal pigment epithelial protein required for the production of the visual chromophore 11-cis-retinal in the visual cycle [12]
We demonstrate the necessity of the conserved histidines and acidic residues in the catalytic mechanism of BCMO1, supporting our hypothesis that these residues are necessary for iron coordination in BCMO1

Summary

EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis—A panel of mutant BCMO1 proteins was made using a previously described pBAD/BCMO1 construct [3] as a template. The culture was allowed to grow at 37 °C to mid-log phase (A600 ϳ 0.6) and was split; one-half was induced with 0.02% (w/v) L-arabinose, whereas the other was not induced Both cultures were grown for another 3 h at 30 °C; the absorbance was measured; and cells were collected by centrifugation at 5000 ϫ g for 20 min. Purified protein was stored in 40% (v/v) glycerol at Ϫ20 °C, and the same batch of purified protein was used as a standard for all ELISAs. 96-Well OptiPlate96FHB (PerkinElmer Life Sciences) plates were coated with 2–100 ng of standard and 0.5– 4-␮l samples/well in 100 ␮l of imidazole-buffered saline (IBS) containing 1 mM tris(2-carboxyethyl)phosphine hydroxychloride and one Complete protease inhibitor tablet/50 ml. The NanoOrange protein quantification kit (Molecular Probes, Inc., Eugene, OR) was used for measurement of diluted protein solutions (Ͻ1 ␮g/ml)

RESULTS

Unstablea

DISCUSSION