The specificity of fatty acid desaturases and hydroxylases

BIOCBIMICA ET BIOPHYSICA ACTA

IO

THE SPECIFICITY OF FATTY ACID DESATURASES THE DEHYDROGENATION

AND HYDROXYLASES

AND HYDROXYLATION

OF MONOENOIC

ACIDS

D. HOWLING+,

L. J. MORRIS,

M. I. GURR

AND

A. T. JAMES

Division of Biochemistry, Biosciences Group, U&ever Colworth House, Sharnbrook, Bedford (Great Britain) (Received

Research

Lahovatory

Colworthl Welwyn,

August r&h, 1971)

SUMMARY

I. The substrate specificities have been studied of (a) a desaturase, which forms a methylene interrupted diene from a monoenoic fatty acid in the alga, C~~~e~a vtdgaris and in a plant seed, Ricinus comvnz~~is; and (b) an hydroxylase, which forms a methylene interrupted monoenoic hydroxy acid from a monoenoic acid in Ricinus. 2. Of a wide variety of monoenoic acids only two types have the structural requirements to act as substrates for either reaction, namely carboxyl-g and s-g monoenes. 3. On the basis of these results it is suggested that two desaturases exist, one recognising monoenoic acids with a double bond at position 9 with respect to the carboxyl group and the other recognising acids with a double bond at position 9 with respect to the methyl group. Oleic acid (do and n-9) would thus be a substrate for both enzymes and is shown to be converted into a dienoic acid (linoleic acid) at least twice as effectively as any other monoenoic acid studied. 4. The evidence also supports the suggestion that there are two hydroxylases with the same structural specificities as the desaturases.

INTRODUCTION

In a recent paper1 we described studies of the conversion of an homologous series of saturated straight chain fatty acids into the corresponding cis-g-monoenoic acids in hen liver, goat mammary gland, Tordopsis bombicola and ChloreUa vulgaris. We also described how the formation of this k-9 double bond was influenced by the presence of branched methyl groups or other &s-double bonds. The results indicated that there is probably a widely distributed desaturase specific for the introduction of a double bond at the AD position irrespective of chain length of the substrate but l Present address: borough, England.

Biochim.

Biophys.

Chemistry

Department,

Acta, 260 (x972) lo-19

Loughborough

University

of Technology,

Lough-

DRHYD~~~NATION having

~~~~~

AND ~~~~OXYLATXON

OF MONOENOIC ACIDS

11

activity E&t Cs, high activity at C;, and C,, and lower activity at C,(,

C,, and GP The spec%city of the position of double bond introduction, the sensitivity toward substituent methyl groups along a large part of the chain, and the precise stereochemical requirement far the dehydrogenation argue strongly, first, for a binding site holding the carboxyl group fixed and precisely locating the 9- and ro-positions of the fatty acyl chain at the active centre; second, for a close enfolding of the substrate acyl chain by the protein. In an earlier papers we had shown that in C. V@G& although ati saturated acids from C,:, to G,, yielded a cGs-As-monoene, only C,,,, &am lengths yielded a diene fA0*13in each case). C,:, gave no Gene. Furthermore C,,, C&, and C,, yielded A’-monoe~es, in addition to the A*-monoenes, but only C,, yielded a diene (AT+). It was therefore relevant to ask the question, what structural features must be possessed by a monoenoic acid in order to be a substrate for the desaturase which introduces a second double bond in the formation of methy~ene-inte~pted dienoic acids*s. In addition, since some plant seeds possess enzymes which introduce a hydroxyl group into fatty acids at positions similar to those occupied by the second double bond in dienoic acids+-@ it was of interest to find out whether the structural requirements of the substrate were also similar to the desaturase. Some preliminary results have already been describedpJ”. The substrates used are shown in Table I. TABLE STRUCTUKR

f OF SUBSTRATE

iWX3CULES

Group I

Fixed ~~~-doubl~ bond distance, variable chrain length (4

Group II

Fixed CH,-double bond distance, variable chain Iengtb (wf

Group 111 Variable double bond position, constant chain length Group IV

EXPERIMENTAL

[r-XJHeptadecanuic {margaric) acid (14.8 rnC~~o~e), &s-g-jr-%)octadecenoic (oleic) acid (40 mC~mmole) and tis, c&3-9,x2-[I-Wjoctadecadienoic flinoleic) acid (5.9 mCfmmo~e) were purchased from the ~a~oche~ca~ Centre. Amersham ~~~~~~d). These were used in the predation of various other Welled substrates as described below. cam-n8[x-l~]Non~ecenoic and cam-Iz-[x-s”C]t>ct~~ccsnoic acids were synthe-

12

D. HOWLING

et al.

sized as described previouslyl. Unlabelled &s-7-octadecenoic and cis-II-octadecenoic acids were the gift of Drs. F. D. Gunstone and I. A. Ismail; q-heptadecynoic acid was a gift from our colleagues at the Unilever Research Laboratory, Vlaardingen (The Netherlands). Other labelled substrates were prepared by methods outlined below. All products were purified as their methyl esters by argentation thin-layer chromatographyl. Their specific activities were approximately the same as the precursors from which they were derived.

(a) cis-9-[r-1C]He$tadecetioic acid. [r-X*C]Heptadecanoic acid (sodium salt) was incubated with the yeast T. bombicoEain its nutrient medium1 at ,room temperature (approx. 21’) for 6 h. The lipids were extracted with chloroform-methanol and the chloroform layer washed with saline solution, The solvent was removed and the lipids transmethylated in meth~ol-benzene-HISS* (20: IO: I, by vol.) to yield fatty acid methyl esters. cis-9- [I-l%]Heptadecenoic acid methyl ester was isolated and purified by preparative argentation thin-layer chromatography followed by preparative gasliquid chromatography. Radiochemical gas-liquid chromatography11 confirmed its activity and radiopurity. acid sodium salt was (b) cis-9-[I-W]N 0%adecenoic acid. [I-%]Nonadecanoic incubated with hen liver microsomes as described previouslyr. Its isolation and purification were exactly as described for heptadecenoic acid in (a) above. Chain elongatiomof available labelled szcbstrates cis-Io-[z-WlNonadecenoic acid was synthesized by chain elongation of cis-o[I-Wloctadecenoic acid via the corresponding alcohol, p-toluene sulphonate and nitrile’. Degradation of zlnlabelled $reczcrsor followed by readdition of a labelled carbon atom (a) cis-8-[I-W]Heptadecenoic acid. Unlabelled cis-8-heptadecenoic acid was prepared from oleic acid by the CRISTOL AND FIRTH 18modification of the Hunsdieker reaction. The labelled carbon atom was then introduced by a second Cristol-Firth degradation followed by elongation back to the C,, compound with labelled KCN as follows : Br2

CH3(CH&CH=CH(CH&$OOH

cc,4

-CH3(CH2)7CH.Br.CH.Br(CH2)6COOH HgO/ 8’2

CH.,tCH,,,CH

=CH(CH&Br

K’.CN/ I CH3(CH2),CH=CH

zn

-

1 CH~CH~,CH.BrCH-8r(CHd68r

CZHPH

DMSO tCH&kN

*

CH@i2)7CH=CH(CH2&j’4COOCH3

(1) KOH/CHJOH (2) HCI i

DMSO=

dimethyl

eulphoxtde

The methyl ester was purified by argentation thin-layer chromatography with 15% ether in light petroleum as solvent and by preparative gas-liquid chromatography. (b) cis-7-[x- YI]Octa&cetioic a& cis-II-[r-W]octadecenoic acids. These acids Biochim. Bioflhys. Acta.

260

(x972)

IO-IQ

DEHYDROGENATION

AND HYDROXYLATION

OF MONOENOIC ACIDS

13

were prepared from the corresponding unlabelled compounds by the method described in (u) above. De ~00~0synthesis from smaller fragments or modi,fication of related acids (a) cis-8-[x-1’C]0ctadecenoic acid. The hydroxyl group of r-chlorod-hydroxyhexane was protected by formation of the z-tetrahydropyranyl ether18 and converted into the iodide by refluxing with NaI. The phosphonium salt was prepared and coupled with decyl aldehyde by a WII-TIG~~ reaction in dimethyl sulphoxide as solvent. Hexadec-6-ene-r-01 was purified by counter current distribution in a light petroleummethanol-water system. The compound was pure as judged by argentation thin-layer chromatography. The chain extension was accomplished via the following reaction scheme’* 16: CH3SO3CI CH3(CH2)~CH=CH(CH~)~CH~H

Pyridine

CH3(CH2,&H=CWWi&COOCH3-HC”CH30H LiAIH, CH301.$,

1 H = CH W4Z)&HgOH

CH3S02CI Pyridine

CCH3(CH,,aCH8CH(CH1)4CH20.50pCH3 KCNiDMSO I CH3tCH&CHtCH(CH2)4CH2CN

-CH3(CH&CH=CHtCH,3&&O*SO&H3 K”CNlDMS0 CHjCH 3 &H.CH(CH&CH2UCN

DMSO=dimethyi

sulphoxide

(b) cis-ro-jr-l~]Octadecenoic acid. Methyl g-heptadecynoate was converted into the corresponding ethylenic compound by diimide reductionltls. This compound was then chain extended by one carbon atom using labelled potassium cyanide in the sequence of reactions described in (a) above. (c) jr-YT]Octadec-9-ynoic acid (stearolic acid). Oleic acid (IOO pC, approx, 0.5 mg) was mixed with IOO mg carrier oleyl alcohol in ether and brominated. The dibromide, without fnrther purification was dehydrobrominated by refluxing with potassium tert.-butoxide in tert.-butanoll’. The product was methylated with diazomethane and purified by thin-layer chromatography to give a 29% yield of methyl [r-W]stearolate. (a) tya~s-Q-[~-l~]Oetadece~o~c acid (elaidic acid). This acid was prepared by isomerization of jr-W]oleic acid with oxides of nitrogen18. Identity of substrates All substrates were checked for identity of the double bond position by permanganate-periodate oxidationlg and determination of the resulting mono and dicarboxylic acids by radiochemical gas-liquid chromatography”. Incubations Desatwahm ahe. Incubation of labelled substrates with C. va&aris and isolation of products have been described previously 1~s.Extent of conversion into dienoic acids was measured by radiochemical gas-liquid chromatography of the isolated fatty acid methyl ester+. Double bond positions were identified by partial diimide reductionl+ of the dienoic acids which had been isolated by argentation thin-layer chro-

D. HOWLINGet at.

I4

matography. The formation of monoenes during the reduction was monitored by gasliquid chromatography and when the yield was maximal, the isomeric monoenes were isoiated by argentation thin-layer chromatography* and oxidized by the VON RUDLOFF~*permanganate-periodate technique. The labelled dicarboxylic acid dimethyl esters were identified by radiochemical gas-liquid chromatography by comparison with the chromatographic behaviour of a standard mixture of dicarboxylic dimethyl esters. Hydroxylation and dtxatwation. Seeds of R&&us commzcniswere first harvested from plants growing in greenhouses 35 days after flowering and checked for their ability to form ricinofeic acid by incubation with [r-W]oleic acidv. Seeds which were capable of about 20% conversion of oleate into ricinoleate were used for experimental incubations. The beans were sliced with a razor blade and three embryos and portions of endosperm were suspended in 0.2 M phosphate buffer. Labelled substrates, dispersed by sonication in water, were added and incubated for 24 h at 30~. Lipids were extracted? and transmethylated. The resulting methyl esters were separated into monoenoic dienoic and hydroxyacid classes by argentation thin-layer chromatography. Plates were developed first in 50% ether in light petroleum for a distance of 8 cm. The solvent was evaporated and the plates were then developed twice in 20% ether in light petroleum. Monoenoic, dienoic and hydroxy acid bands were scraped from the plates and the radioactivity of each band analysed directly by scintillation spectrometry in 0.4% PPO in toluene. In parallel incubations the chain length of the eluted monoenoic hydroxy acid methyl esters was checked by ra~ochemic~ gas-liquid chromatoTABLE II THE CONVERSION 0F MONOENOIC To DIENOIC FATTY

ACIDS z3Y Clilove&z vulgaris

o/oDiuect

Precursor

Double bond position

conversion* Group I cis-g-Hexadecenoic cis-g-Hepadecenoic cis-g-Octadecenoic cis-g-Nonadecenoic

21.0

(n-7) * * (n-8) (n-9) (n-10)

37.4 79.0 25.0

Group II cis-7-Hexadecenoic (n-g) c&=&Heptadecenoic (n-9) ck-g-Octadecenoic (n-g) cis-ro-Nonadecenoic (n-g)

36.0 26.0 79.0 24.1

Group III cis-7-Octadecenoic (n-s I) cis-8-Octadecenoic (lt-IO) cis-IO-Octadecenoic (g-8) k-1 I- ?ctadecenoic (n-7) cis-12-Octadecenoic (w-6)


Group IV trans-g-Octadecenoic (n-g) g-Octadecynoic h-g, I S-Nonadecadienoic (r-9) * * *


9-10. g-10, g-10, g-10,

12-13 12-13 12-13 I*-I3

7-8, IO-II (s-6, n-g) 8-g. II-12 (n-6, n-g) g-10, 12-13 (n-6, s-9) I~II, 13-14 (n-6,19-9)

48.0

o-10,

12-13

72.7

g-10,

12-13, 18-19

* In some cases breakdown and resynthesis de nova occurred, see RESULTS and DISCUSSION. ** Presumed precursor from palmitic acid, see RESULTS and DISCUSSION. *** Presumed precursor from cis-r8-nonadecenoic acid, see RESULTS and DISCUSSION. The figure of 72.7% represents radioactivity in the monoene and diene fraction. Individual %gureS are not available.

Biochim.

Biophys.

Ada,

260 (1972) IO-19

DRHY~RO~~NATION

AND HYRROXYLATION

15

OF MONOENOIC ACIDS

graphy and the position of the hydroxyl group dete~n~ fh-st by reduction to the saturated analogue followed by oxidation with CrO, in glacial acetic acid. The resulting labelled dicarboxylic acids were converted into methyl esters, purified by thinlayer chromatography and identified by radiochemical gas-liquid chromatography. RESULTS Cowersion

of mnoenoic

ido

diem&c acids

C. vsdgaris cells and castor bean tissue all monoenoic acids of Group I (carboxyl-g monoenes) were converted into &,cks-g,rz-dienes in yields of zr-37% (Tables II and III). Similarly all Group II (n-g) monoenes were converted into c&,&s1x1 both

TABLE

III

CONVERSION

OF MONOENOIC

FATTY

ACIDS

TO DIENOIC

FATTY

0/oDir& coneersim * i& dim& acid

PR%XW%3Y

ACIDS

BY

Ricileus CO?Pt?BU?&iS SEEDS

Dm&ie baband posifioa in jw13radtsc#

Group I cis-Q-I-hxadecenoic (n-7)** cis-g-Heptadecenoic (n-8) cis-g-Octadecenoic (n-g) cis-g-Nonadecenoic (n-IO)

6.7 6.8 9.2 4.5

g-10, g-10, Fro, g-10,

Group II cis-&Heptadecenoic (a-g} cis-g-Octadecenoic (n-g) c~s-1o-Nouad~enoic (n-g)

2.8 9.2 2.0

8-9, IX-I? (n-6, n-g) g-10, 12-13 (n-6, n-g) IO-II, 13-14 (~6, n-9)

Group III cis-Mktadecenoic cis-ro-Octadecenoic

&-IO) (n-8)

*. * * See corresponding

<1
12-13 12-13 12-13 x2-13

-

footnotes to Table 11.

(n-Q-g)-dienes in yields of 24-36%. Oleic acid, which is both a carboxyl-g and an n-g monoene was converted to roughly twice the extent of any other monoene in Groups I and II. However, in all cases except two, where the initial double bond was neither in the carboxy-g nor the s-g position (Group III monoenes) there was no detectable diene formation. The two exceptions to this rule were cis-rz-octadecenoic acid, which was converted into cks,cis-g,rz-octadecadienoic acid (linoleic acid) in 48% yield, and r8-nonade~noic acid, which was converted into c~s-g,r8~nonadeca~enoic and cti,eksg,x2,18-nonde~t~enoic acids. In two cases (cis-7 and us-g-hexadecenoi~ acids) the incubated precursor was not the monoenoic acid but the corresponding labelled saturated acid, palm& acid. It was previously established that pahnitic acid can be desaturated by Chlorella to both d’ and d*-C,,-monoenes* and it is inferred that these isomeric monoenes are the precursors for the cis,&-7,ro-diene and n’s,&+g,xz-diene, respectively. As implied above, modification of a cis-g-monoene by introduction of a terminal double bond (cis-g,r8-nonadecadieuoic acid) did not affect its ability to act as a substrate for the desaturase, but rn~~~tion of the first double bond by converting into a &ass double bond (elaidic acid) or into an acetylenic bond (stearolic acid) made the compounds completely inactive as desaturase substrates. Similar results were obtained for the hy~oxylatio~ reaction catalysed by R. c~~~~~~s seeds. Group I substrates were hydroxylated at the xz position (Table IV)

16

D. HOWLING et a,?.

while Group II substrates were hydroxylated at the n-6 position. Again, oleic acid was hydroxylated to twice the extent of any other substrate. Compounds of Groups III and IV were not hydroxylated. TABLE THE

IV

CONVERSION

Ricinus communis

OF

MONOENOIC

(CASTOR)

SEED

Group I cis-g-Hexadecenoic (n-7) * * cis-g-Heptadecenoic (n-8) cls-g-Octadecenoic (n-g) cis-g-Nonadecenoic (n-zo) Group II ch-SHeptadecenoic cis-g-Octadecenoic

12

(n-10)

0.9-1.9

(n-7)

footnotes

FATTY

ACIDS

BY

THE

Refs. 6, 7

I2 12

II 12

(n-6) (~-6)

I3 (n-6) II


Group IV tvans-g-Octadecenoic ** * * See corresponding

HYDROXY-MONOENOIC

I2

5.6

(n-8)

TO

9.0 ‘7.4 4.8

17.4 4.3

(n-9)

cis-ro-Octadecenoic &s-r I -0ctadecenoic

ACIDS

6.2

(n-9) (n-9)

cis-ro-Nonadecenoic Group III ck-B-Ochdecenoic

FATTY SYSTEM

0

-

Ref. 6

0

-

Ref. 6

to Table II.

DISCUSSION

The aim of this study was to elucidate the “substrate recognition system” (or specificity) of the diene-forming desaturase. We shall therefore outline the possible ways in which the enzyme might recognize its substrate, based on the knowledge derived from studying monoene-forming desaturases (see INTRODUCTION and ref. I) and compare these hypotheses with the present experimental results. The possibilities are : (x) Alignment of the carboxyl group (or a functionalised version of it such as the acyl-S-CoA or acyl-S-acyl carrier protein thiol ester) at a fixed point thus bringing the active centre of the enzyme to a fixed distance along the chain from the carboxyl group. This means of locating the new double bond would thus be independent of the position of the existing double bond. (z) Recognition of the terminal methyl group and alignment of the active centre at a fixed distance along the chain. As in the first possibility this means of locating the new double bond would also be independent of the existing double bond position. (3) Alignment of the existing double bond alone with some specific binding site so bringing the active centre to a fixed position with respect to the double bond. (4) Recognition of a gap of specific size between the carboxyl group (or some functionalised version of it) and the existing double bond, aligning the active centre with respect to both. (5) Recognition of a gap of specific size between the terminal methyl group and an existing double bond, aligning the active centre at a fixed distance from both. Biockim.Biophys.

Acta,

260

(1972)

Ic--Ig

~EH~DR~ENATION

AND HYDROXYLATION

OF ~ONOENOI~

ACIDS

17

The validity of each of these hypotheses can now be tested by comparison with the experimental results outlined above and presented in Tables II, III and IV.

Possibility I The new double bond (or hydroxyl group) introduced in this case would be always at a fixed position from the carboxyl group and so the cis-g acids should give rise to 9,rz-dienoic acids (as they do, Tables II and III) and acids with double bonds at other positions should also give rise to Ala-dienoic acids in which the double bonds are not methylene interrupted (which they do not, Table III). This hypothesis is therefore invalid.

In no case is the newly introduced double bond at a fixed methyl distance unaffected by an existing double bond, so that this hypothesis also is untenable.

Possibility 3 Here the new double bond or hydroxyl group should be at a fixed position with respect to any double bond position. The tables show that neither &s-7-&8-,&10nor cis-II-octadecenoic acids are enzymic substrates, so that this hypothesis is also untenable.

Possibility 4 In this case only specific carboxyl-distant monoenoic acids would be substrates. This is true for all the k-g acids but is contra-located by the c&-7-hexadecenoic, cz’s-8-heptadecenoic and ck-ro-nonadecenoic acids which give rise to products with groups situated one CH, group away from the existing double bond.

Possibility 5 Here the new groups should be specified with respect to an existing double bond at a fixed distance from the methyl group. The series of %g acids all give rise to n-6, n-g dienoic acids and so they obey this pattern. The As acids, irrespective of chain length (i.e. with variable n-x position) also form dienoic acids with the new double bond at the Afa position, so those substrates do not fit this hypothesis. Clearly there are two groups of enzyme substrates, the A* acids and the “9 acids and the two structural requirements cannot be fitted to a single enzyme. We are therefore forced to the conclusion that two enzymes exist, one dealing with the A* acids of variable chain length and the other with n-9 acids also of variable chain length. It is worth emphasizing that oleic acid is the only substrate that has both the A@and n-9 structure and examination of Tables II and IV shows that oleic acid is converted into the dienoic or hydroxylated acid roughly twice as effectively as any other acid. This could be expected if it, and it alone, were the substrate for two enzymes. The possibility that there is one enzyme which has a fair degree of latitude in the positioning of the acyl chain at the active site, and that this could account for desaturation of Group II substrates, is virtually eliminated by the fact that &s-8- and cis-rooctadecenoic acids are completely inactive as substrates. The double bonds here are only one carbon atom away from that of the natural substrate, yet there is no desaturation. This indicates extremely rigid positional specificity. In some experiments, dienes

x8

D. HC3WEJNG & &.

were apparently formed after incubations of Chorella with bus-xo-~tadecenQic acid. Radiochemical gas-liquid c~omato~phy showed that an appreciable proportion of the radioactivity (13%) was in methyl esters of shorter chain length than the precursor suggesting breakdown of the substrate by @-oxidation, followed by re-synthesis. The diene fraction in this experiment was oxidized directly with periodate-permanganate. The only labelled dicarboxylic acids were C,- (arising from cis,cis-7,x0-r6: z) and C, (arising from cis,c&-9~2-16: 2 and c&+&s-g,rz-18: a). No C, dicarboxylic acid was detected, proving that there had been no direct desaturation of cis-xo-octadecenoic acid. In only one case was there any indication that this rigid specificity did not obtain. Cis-8-octadecenoic acid gave rise to a trace of Ix-hydroxy-8-octadecenoate {the expected product of direct hydroxylation) in addition to a larger amo~t (g-17%) of xz-hy~oxy-g-octadec~noate (formed by breakdown and resynthesis). However, the radioactivity in the I I-hydroxy compound isolated by preparative gas-liquid chromatography was only twice the back5ound level and was probably not significant. The apparently anomalous desatnration of the Group III monoene &-x2octadecenoic acid has been discussed previously lfl”. It seems likely that the desaturase concerned in this conversion is the stearate (“monoene-forming”) desaturase, in view of the position at which the double bond is inserted (A@)and the fact that the reaction may be catalysed by an animal desaturase (in hen liver microsomes) which does not normally form linoleic acid. A more detailed investigation of the desaturation of cisrz-octadecenoic acid by a number of different animal species and tissues will be presented in a future publication. The complexity of naming the desaturase enzymes makes a shorthand nomenclature convenient for discussion. Hence we suggest naming the desaturase acting on saturated acids and de&&g the double bond position with respect to the carboxyl group as desatnrase IA. The enzyme converting monoenoic acids into dienoic acids that inserts the double bond in a position defined also with respect to the carboxyl group could then be called desaturase IIA. The third enzyme, suggested by our present results, that recognises double bond position with respect to the terminal methyl group could thus be called desaturase 110 (see Table V). There are therefore clear similarities between desaturase Id and desaturase HA. These enzymes obviously require a substrate that allows strong interaction between some group on the protein and a functional&d version of the carboxyl group of the fatty acid (such as the acyl-S-CoA or acyl-S-acyl carrier protein thiol ester) in order to fulfil their positional speci~cities (Table V). The enzyme IIw, however, does not involve such an interaction since substrate specificity is conferred by distance from the methyl end, Other work in this laboratory has snggested that at least some oleic acid in Chlorella may be desatnrated in the form of oleoyl phosphatidyl choline giving rise directly to linoleoyl phosphatidyl choline 4~10.An enzyme of the 110 type might thus provide a satisfactory means of desaturating lipid-linked acyl groups (Table V). Such a concept, too, is consistent with, but not proved by, the observed distribution of C,, and C,, saturated, monoenoic, dienoic and trienoic acids on the I-,and a-positions of Chloreila galactosyl diglyceridePl. The work of SCWULZ AND LYNEN~~ with desaturase IA and of VIJAY AND STUXPF~* with desaturase IIA shows clearly that CoASH is not liberated during the reaction, There is therefore no ~ke~hood of these two desaturases involving an acyltransferase that acylates the enzyme and liberates free CoASH. These results do not

DEHYDROGENATION

TABLE

AND HYDROXYL-ATION

OF MONOENOIC

ACIDS

19

V

SUBSTRATE RECOGNITION PATTERNS OFDIFFERENT Substrate recognition pa&*n HOOC(CH,),CH,.

CH,(CH,),CH,

DESATURASES

Suggested substrate

Reaction catalysed

Ensyme nomenclatwe

Acyl-S-CoA

Saturated C14-Cl8

Desaturase Id

Ol-

HOOC(CH,),CH=CH-(CH,)&H,

HOOC(CH,),,CH=CH(CH,),CH,

Acyl-S-acyl carrier protein Acyl-S-CoA or Acyl-S-acyl carrier protein Acyl-0-phospholipid

J-

AD-monoene Oleic

Desaturase

4 linoleic Oleic

Desaturase 11~

IIA

4 linoleic

conflict with our views since we have never argued for a singularity of mechanisms. Although the substrate requirements for hydroxylation appear to be analogous to those of the desaturases, and therefore two enzymes appear to be involved, we have as yet no parallel evidence regarding the hydroxylation of lipid linked acyl chains. ACKNOWLEDGEMENTS

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