Desaturation of a fatty acid first involves the enzymatic removal of a hydrogen from a methylene group in an acyl chain, a highly energy-demanding step that requires an activated oxygen intermediate. Two types of desaturases have been identified, one soluble and the other membrane-bound, that have different consensus motifs. Database searching for these motifs reveals that these enzymes belong to two distinct multifunctional classes, each of which includes desaturases, hydroxylases, and epoxidases that act on fatty acids or other substrates. The soluble class has a consensus motif consisting of carboxylates and histidines that coordinate an active site diiron cluster. The integral membrane class contains a different consensus motif composed of histidines. Biochemical and structural similarities between the integral membrane enzymes suggest that this class also uses a diiron cluster for catalysis. Soluble and membrane enzymes have been successfully re-engineered for substrate specificity and reaction outcome. It is anticipated that rational design of these enzymes will result in new and desired activities that may form the basis for improved oil crops.

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)

The three‐dimensional structure of recombinant homodimeric delta9 stearoyl‐acyl carrier protein desaturase, the archetype of the soluble plant fatty acid desaturases that convert saturated to unsaturated fatty acids, has been determined by protein crystallographic methods to a resolution of 2.4 angstroms. The structure was solved by a combination of single isomorphous replacement, anomalous contribution from the iron atoms to the native diffraction data and 6‐fold non‐crystallographic symmetry averaging. The 363 amino acid monomer consists of a single domain of 11 alpha‐helices. Nine of these form an antiparallel helix bundle. The enzyme subunit contains a di‐iron centre, with ligands from four of the alpha‐helices in the helix bundle. The iron ions are bound in a highly symmetric environment, with one of the irons forming interactions with the side chains of E196 and H232 and the second iron with the side chains of E105 and H146. Two additional glutamic acid side chains, from E143 and E229, are within coordination distance to both iron ions. A water molecule is found within the second coordination sphere from the iron atoms. The lack of electron density corresponding to a mu‐oxo bridge, and the long (4.2 angstroms) distance between the iron ions suggests that this probably represents the diferrous form of the enzyme. A deep channel which probably binds the fatty acid extends from the surface into the interior of the enzyme. Modelling of the substrate, stearic acid, into this channel places the delta9 carbon atom in the vicinity of one of the iron ions.

The subfamily of the Lemnoideae belongs to a different order than other monocotyledonous species that have been sequenced and comprises aquatic plants that grow rapidly on the water surface. Here we select Spirodela polyrhiza for whole-genome sequencing. We show that Spirodela has a genome with no signs of recent retrotranspositions but signatures of two ancient whole-genome duplications, possibly 95 million years ago (mya), older than those in Arabidopsis and rice. Its genome has only 19,623 predicted protein-coding genes, which is 28% less than the dicotyledonous Arabidopsis thaliana and 50% less than monocotyledonous rice. We propose that at least in part, the neotenous reduction of these aquatic plants is based on readjusted copy numbers of promoters and repressors of the juvenile-to-adult transition. The Spirodela genome, along with its unique biology and physiology, will stimulate new insights into environmental adaptation, ecology, evolution and plant development, and will be instrumental for future bioenergy applications.

Salicylic acid (SA) plays an important role in activating various plant defense responses, including expression of the pathogenesisrelated (PR) genes and systemic acquired resistance. A critical positive regulator of the SA signaling pathway in Arabidopsis is encoded by the NPR1 gene. However, there is growing evidence that NPR1-independent pathways can also activate PR expression and disease resistance. To elucidate the components associated with NPR1-independent defense signaling, we isolated a suppressor of the npr1-5 allele, designated ssi2. The recessive ssi2 mutation confers constitutive PR gene expression, spontaneous lesion formation, and enhanced resistance to Peronospora parasitica.

Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) malesterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development. Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots.

The stearoyl-ACP delta 9 desaturase from plants is a new example of a growing number of proteins that contain oxo- or hydroxo-bridged diiron clusters. On the basis of differences in primary sequence motifs providing the cluster ligands and upon structural differences elucidated by X-ray crystallography, we now propose that the presently known, soluble diiron-oxo proteins can be grouped into two classes, I and II. Class I contains hemerythrin, myohemerythrin, and, possibly, purple acid phosphatase. Class II contains ribonucleotide reductases, bacterial hydrocarbon hydroxylases (methane monooxygenase, toluene-4-monooxygenase, and phenol hydroxylase), rubrerythrin, and stearoyl-ACP desaturases. Through the use of resonance Raman spectroscopy, we have detected symmetric (vs = 519 cm-1) and asymmetric (vas = 747 cm-1) vibrational modes in the castor stearoyl-ACP delta 9 desaturase, which are typical of oxo-bridged diiron clusters. These frequencies shift by -18 and -34 cm-1, respectively, in H218O, proving that the bridging ligand is readily exchangeable with solvent (t1/2 = 7 min). Calculation of an approximately 123 degrees Fe-O-Fe angle from the position of vs and vas and from the 18O-dependent shift in these frequencies suggests that the diiron-oxo cluster in the desaturase is triply bridged in the diferric state. In the diferrous state, the two iron sites of the cluster are structurally inequivalent, as shown by differential temperature dependence of the Mössbauer quadrupole splittings. For the class II diiron-oxo proteins, primary sequence alignments reveal conserved amino acid residues which act as iron cluster ligands, participate in a hydrogen-bonding network, and are potentially involved in O2 binding and activation. Based on this conservation, a structural model for the stearoyl-ACP delta 9 desaturase active site is proposed that has strong similarity to both ribonucleotide reductase and methane monooxygenase. However, after single turnover of the diferous state with 18O2, 18O is not detected in the oxo bridge of the castor desaturase. This is in contrast to the outcome observed for ribonucleotide reductase, suggesting the desaturase and ribonucleotide reductase differ in certain aspects of their respective O2-activation reactions.

Male Sterile2 (MS2) is predicted to encode a fatty acid reductase required for pollen wall development in Arabidopsis (Arabidopsis thaliana). Transient expression of MS2 in tobacco (Nicotiana benthamiana) leaves resulted in the accumulation of significant levels of C16 and C18 fatty alcohols. Expression of MS2 fused with green fluorescent protein revealed that an amino-terminal transit peptide targets the MS2 to plastids. The plastidial localization of MS2 is biologically important because genetic complementation of MS2 in ms2 homozygous plants was dependent on the presence of its amino-terminal transit peptide or that of the Rubisco small subunit protein amino-terminal transit peptide. In addition, two domains, NAD(P)H-binding domain and sterile domain, conserved in MS2 and its homologs were also shown to be essential for MS2 function in pollen exine development by genetic complementation testing. Direct biochemical analysis revealed that purified recombinant MS2 enzyme is able to convert palmitoyl-Acyl Carrier Protein to the corresponding C16:0 alcohol with NAD(P)H as the preferred electron donor. Using optimized reaction conditions (i.e. at pH 6.0 and 30°C), MS2 exhibits a K(m) for 16:0-Acyl Carrier Protein of 23.3 ± 4.0 μm, a V(max) of 38.3 ± 4.5 nmol mg⁻¹ min⁻¹, and a catalytic efficiency/K(m) of 1,873 M⁻¹ s⁻¹. Based on the high homology of MS2 to other characterized fatty acid reductases, it was surprising that MS2 showed no activity against palmitoyl- or other acyl-coenzyme A; however, this is consistent with its plastidial localization. In summary, genetic and biochemical evidence demonstrate an MS2-mediated conserved plastidial pathway for the production of fatty alcohols that are essential for pollen wall biosynthesis in Arabidopsis.