Eight Histidine Residues Are Catalytically Essential in a Membrane-Associated Iron Enzyme, Stearoyl-CoA Desaturase, and Are Conserved in Alkane Hydroxylase and Xylene Monooxygenase

Abstract

The eukaryotic fatty acid desaturases are iron-containing enzymes that catalyze the NAD-(P)H- and O2-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.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of Rv3230c as the NADPH Oxidoreductase of a Two-Protein DesA3 Acyl-CoA Desaturase inMycobacterium tuberculosisH37Rv

“…Kyte-Doolittle analysis of these other proteins revealed the presence of multiple hydrophobic sequences that may span the cellular membrane (26). Likewise, previous Kyte-Doolittle hydropathy analysis suggested that DesA3 might contain up to three hydrophobic domains (5).…”

“…One product of this reaction, oleic acid, is an essential constituent of mycobacterial membrane phospholipids and triglycerides (5,(23)(24)(25). Thus DesA3 is suggested to be a target of isoxyl (also known as thiocarlide), an anti-tuberculosis drug that inhibits the synthesis of oleic and mycolic acids (5).DesA3 shares the conserved eight His motif found in membrane acyl-lipid and acyl-CoA desaturases and in the related bacterial membrane enzymes alkane hydroxylase and xylene monooxygenase (5,(26)(27)(28). By this assessment, DesA3 is likely to be an integral membrane diiron enzyme.…”

Structural Control of Chemoselectivity, Stereoselectivity, and Substrate Specificity in Membrane-Bound Fatty Acid Acetylenases and Desaturases

“…The positions of the 11 conserved differences determined from the alignment are shown in Figure 2, overlaid on a topological model established for membrane-bound desaturases (11). It is notable that all but 2 of the positions are predicted to be on the cytosolic side of the endoplasmic reticulum and 7 out of the 11 positions are within 12 amino acids of one of three His boxes which are thought to be involved in binding a diiron cluster at the active site of these enzymes.…”

The convergent evolution of defensive polyacetylenic fatty acid biosynthesis genes in soldier beetles

“…Full-length sequence of what was likely to be the conserved ∆9 desaturase (CL11, JQ619632) was also obtained. All encoded protein sequences were confirmed as desaturases, as they contained the eight conserved histidine residues that form the 'histidine boxes' that are essential for desaturase activity 21 , were of length between 327-354 amino acids and were aligned with the desaturases of mammals and insects that utilize CoA-linked substrates ( Supplementary Fig. S2).…”