Peter H. Buist

Fatty acid desaturation: variations on an oxidative theme.

Significant progress in our understanding of the mechanism of fatty acid desaturation has been achieved. The site of initial oxidation has been determined for several membrane-bound desaturases and a common cryptoregiochemical theme has been revealed. The results of several studies, including a detailed analysis of a soluble plant desaturase system, point to a close mechanistic relationship between dehydrogenation and hydroxylation pathways.

(S)-α-methoxyphenyl acetic acid : a new NMR chiral shift reagent for the stereochemical analysis of sulfoxides

Abstract The use of ( S )- α -methoxyphenyl acetic acid (MPAA) as a general chiral 1 H NMR shift reagent for the stereochemical analysis of sulfoxides is demonstrated. Using this methodology, both the enantiomeric purity and the absolute configuration of a wide variety of sulfoxides can be determined.

Use of sulfur as an oxidant detector

Abstract The oxidation of a number of methyl thiastearates has been examined as a function of sulfur position using the yeast, Saccharomyces cerevisiae .

Revealing the catalytic potential of an acyl-ACP desaturase: Tandem selective oxidation of saturated fatty acids

It is estimated that plants contain thousands of fatty acid structures, many of which arise by the action of membrane-bound desaturases and desaturase-like enzymes. The details of “unusual” e.g., hydroxyl or conjugated, fatty acid formation remain elusive, because these enzymes await structural characterization. However, soluble plant acyl-ACP (acyl carrier protein) desaturases have been studied in far greater detail but typically only catalyze desaturation (dehydrogenation) reactions. We describe a mutant of the castor acyl-ACP desaturase (T117R/G188L/D280K) that converts stearoyl-ACP into the allylic alcohol trans-isomer (E)-10-18:1-9-OH via a cis isomer (Z)-9-18:1 intermediate. The use of regiospecifically deuterated substrates shows that the conversion of (Z)-9-18:1 substrate to (E)-10-18:1-9-OH product proceeds via hydrogen abstraction at C-11 and highly regioselective hydroxylation (>97%) at C-9. 18O-labeling studies show that the hydroxyl oxygen in the reaction product is exclusively derived from molecular oxygen. The mutant enzyme converts (E)-9-18:1-ACP into two major products, (Z)-10-18:1-9-OH and the conjugated linolenic acid isomer, (E)-9-(Z)-11-18:2. The observed product profiles can be rationalized by differences in substrate binding as dictated by the curvature of substrate channel at the active site. That three amino acid substitutions, remote from the diiron active site, expand the range of reaction outcomes to mimic some of those associated with the membrane-bound desaturase family underscores the latent potential of O2-dependent nonheme diiron enzymes to mediate a diversity of functionalization chemistry. In summary, this study contributes detailed mechanistic insights into factors that govern the highly selective production of unusual fatty acids.

Exotic biomodification of fatty acids

Many biotransformations of mid- to long chain fatty acyl derivatives are intrinsically interesting because of their high selectivity and novel mechanisms. These include one carbon transfer, hydration, isomerization, hydrogenation, ladderane and hydrocarbon formation, thiolation and various oxidative transformations such as epoxidation, hydroxylation and desaturation. In addition, hydroperoxidation of polyunsaturated fatty acids leads to a diverse array of bioactive compounds. The bioorganic aspects of selected reactions will be highlighted in this review; 210 references are cited.

Substrate specificity, regioselectivity and cryptoregiochemistry of plant and animal ω-3 fatty acid desaturases

In order to define the substrate requirements, regiochemistry and cryptoregiochemistry of the omega-3 fatty acid desaturases involved in polyunsaturated fatty acid formation, the genes Fad3 and fat-1 from Brassica napus and the nematode Caenorhabditis elegans respectively were expressed in bakers yeast (Saccharomyces cerevisiae). Various fatty acids, including deuterium-labelled thia-fatty acids, were supplied to growing cultures of transformed yeast. The results from GC-MS analysis of the desaturated products indicate that both the plant and animal desaturases act on unsaturated substrates of 16-20 carbons with a preference for omega-6-unsaturated fatty acids. The regioselectivities of both enzymes were confirmed to be that of omega-3 desaturases. The primary deuterium kinetic isotope effects at C-15 and C-16 of a C(18) fatty acid analogue were measured via competitive incubation experiments. Whereas k(H)/k(D) at the omega-3 position was shown to be large, essentially no kinetic isotope effect at the omega-2 position was observed for the plant or the nematode enzymes. These results indicate that omega-3 desaturation is initiated by an energetically difficult C-H bond cleavage at the carbon closer to the carboxyl terminus. These results will be discussed in the context of a general model relating the structure and function of membrane-bound fatty acid desaturases featuring different regioselectivities.

Stereochemical analysis of d-glucopyranosyl-sulfoxides via a combined NMR, molecular modeling and X-ray crystallographic approach

Abstract ( S )-α-Methoxyphenylacetic acid (MPAA) was used as an NMR shift reagent in combination with molecular modeling to predict the absolute configuration of a representative epimeric pair of glucopyranosyl sulfoxides. The correctness of this assignment was confirmed by X-ray crystallographic examination of one of the epimers, 3a1 . The crystal structure of ethyl 2,3-di- O -acetyl-4,6- O -benzylidene-1-thio-β- d -glucopyranoside S -oxide monohydrate 3a1 was solved by direct methods and was shown to bear the ( R )-configuration at the sulfinyl center in accordance with our prediction. Furthermore, the conformation of 3a1 in the solid state was found to be remarkably similar to that predicted by molecular mechanics calculations.

Highly chemo-, regio-, and stereoselective introduction of a cis-double bond into a thia-analogue of stearic acid

Abstract Replacement of a methylene group by a sulfur atom at the 5-position of stearic acid does not prevent the introduction of a cis -double bond at the 9-,10-positions.

Use of sulfur as a chemical connector

Abstract Replacement of a methylene group by a sulfur atom at the 6-position of oleic acid does not prevent biomethylenation at the olefinic bond.

Bioorganic chemistry of plant lipid desaturation

The desaturation of long-chain fatty acids is a ubiquitous biotransformation which plays a critical role in the biosynthesis of plant lipids. Species-specific variations lead to unusual fatty acid signatures. Of particular interest is the unique ability of desaturases to oxidize unactivated hydrocarbon chains in such a chemo-, regio- and stereoselective manner. As part of ongoing research into the structure/activity relationships of this large class of enzymes, useful mechanistic tools have been developed to probe the active site. Recently a combination of stereo- and regiospecific deuterium, sulfur and fluorine labelling has been used to study the mechanism of a soluble plant Δ9 desaturase. The study of several membrane-bound desaturases has led to the conclusion that the dehydrogenation (desaturation) process is initiated by a kinetically important hydrogen activation step at the carbon of the incipient double bond which is closest to the acyl terminus of the fatty acid chain. A detailed analysis of a related 1,4-desaturation process has also been carried out.