8 HISTIDINE-RESIDUES ARE CATALYTICALLY ESSENTIAL IN A MEMBRANE-ASSOCIATED IRON ENZYME, STEAROYL-COA DESATURASE, AND ARE CONSERVED IN ALKANE HYDROXYLASE AND XYLENE MONOOXYGENASE

The eukaryotic fatty acid desaturases are iron-containing enzymes that catalyze the NAD-(P)H- and O-2-dependent introduction of double bonds into methylene-interrupted fatty acyl chains. Examination of deduced amino acid sequences for the membrane desaturases from mammals, fungi, insects, higher plants, and cyanobacteria has revealed three regions of conserved primary sequence containing HX(3 ?(or 4)) H,HX((2 or 3)) HH, and HX((2 or 3)) HH. This motif is also present in the bacterial membrane enzymes alkane hydroxylase (omega-hydroxylase) and xylene monooxygenase. Hydropathy analyses indicate that these enzymes contain up to three long hydrophobic domains which would be long enough to span the membrane bilayer twice. The conserved His-containing regions have a consistent positioning with respect to these potential membrane spanning domains. Taken together, these observations suggest that the membrane fatty acid desaturases and hydrocarbon hydroxylases have a related protein fold, possibly arising from a common ancestral origin. In order to examine the functional role of these conserved His residues, we have made use of the ability of the rat Delta(9) desaturase gene to complement a yeast strain deficient in the Delta(9) desaturase gene function (ole1). By site-directed mutagenesis, eight conserved His residues in the rat Delta(9) desaturase were individually converted to Ala. Each His --> Ala mutation failed to complement the yeast ole1 mutant. In contrast, mutation of three nonconserved flanking His residues or a partially conserved Arg residue within the conserved motif to Ala allowed for complementation of the ole1 phenotype. Western blot analysis showed that steady-state expression levels were equivalent for the wild-type rat desaturase and for all mutants, suggesting that the conserved His residues are essential for catalytic function. One role for these His residues would be to act as ligands for the iron atom(s) contained in these enzymes. On the basis of these results and the presently available spectroscopic data, we recognize the possibility that the membrane desaturases, alkane hydroxylase, and xylene monooxygenase contain a new structural type of diiron center.