A fatty acid synthetase from Corynebacterium diphtheriae has been purified to a specific activity of 450 nmoles of malonyl coenzyme A incorporated per min per mg. The enzyme is optimally active in 0.5 M phosphate buffer. C. diphtheriae appears to be the most primitive organism having a multienzyme complex for fatty acid synthesis

Knoche, Herman W.

Koths, Kirston E.

English

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Biochemistry -- Faculty Publications Biochemistry, Department of
1973
Characterization of a Fatty Acid Synthetase from
Corynebacterium diphtheriae
Herman W. Knoche
Harvard University
Kirston E. Koths
Harvard University
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Knoche, Herman W. and Koths, Kirston E., "Characterization of a Fatty Acid Synthetase from Corynebacterium diphtheriae" (1973).
Biochemistry -- Faculty Publications. 263.
http://digitalcommons.unl.edu/biochemfacpub/263
THE JOURNAL OF BIOLOGICAL CHEMISTRY 
Vol. 248, No. 10, Issue of May 25, pp. 3517-3519, 1973 
Printed in U.S.A. 
Characterization of a Fatty Acid Synthetase 
from Corynebacterium diphtheriae 
(Reeeived for publication, October 4, 1972) 
HERMAN W. KNOCHE'" AC\'D KIRSTO;-;- E. KonIs 
From The James Bryant Conant Laboratories, Harvard University, Cambridge, Massachusetts 02138 
SUMMARY 
A fatty acid synthetase from Corynebacterium diphtheriae 
has been purified to a specific activity of 450 nmoles of malo-
nyl coenzyme A incorporated per min per mg. The enzyme 
is optimally active in 0.5 M phosphate buffer. C. diphtheriae 
appears to be the most primitive organism having a multi-
enzyme complex for fatty acid synthesis. 
The fatty acid synthetases of animals (1-4), yeast (5), and 
other fungi (6) are multienzyme complexes which have been 
called type I synthetases (7). Type II sYllthetases, which re-
quire added acyl carrier protein for activity, have been observed 
in plants (8, 9) and bacteria (10, ll). Exceptions to this gen-
eralization have been reported. Jfycobacteriurn phlei, one of the 
more advanced procaryotes, has a type I synthetase (7), and 
green Euglena gracilis appears to have both types of fatty acid 
synthetases (12). 
Fatty acid synthesis in Corynebacterium diphtheriae was in-
vestigatcd to determine whether M. phlei wa::; a singular example 
of a proearyote having a type I synthetase and whether phylo-
genetic relationships between organisms and the types of fatty 
acid synthetases exist. 
EXPERIMENTAL PRoC1,Dum; AND RESULTS 
Enzyme Assay-Unless stated differently, the aHsay mixture 
had a volume of 0.5 ml and contained 250 ;tl1lOles of potassium 
phosphate (pH 7.3, optimum pH), 15 nmoles of TPNH, 15 
nmoles of DPNH, 1 nmole of FMN, 50 nmoles of acetyl-CoA, 
10 nmoles of [2-14C]malonyl-CoA (2 MCi per Mmole), 3 Mmoles of 
dithiothreitol, 50 Mg of bovine serum albumin, and 0.11 Mg of 
enzyme protein. Activity, incorporation of radioactivity from 
[2-!4Clmalonyl-CoA into fatty acids, was determined as described 
previously (13). 
Culture-A culture of C. diphtheriae, strain C-7, R ( , tox( - ), 
was a gift from Dr. A. M. Papperheimer of the Biological Lab-
oratories, Harvard University. The orgauisllls, grown at 30° 
in a medium containing 10 g per liter of yeast extract and 15 g per 
* Recipient of Fellowship 1 F03 HE51372-01 from the Depart-
ment of Health, EdLlCation, and Welfare, National Institutes of 
Health. Present address, Department of Biochemistry and Nu-
trition, East Campus, University of Nebraska, Lincoln, Nebraska 
68503. 
liter of maltose (65%, Tech gradp) were harvested in the late log 
phase of growth.! 
Enzyme PurificationL-The washed cells were suspended in 
0.55 M potassium-phosphate buffer, pH 8.0, which also contained 
1 mM EDT A and 1 mM dithiothreitol, and were disrupted with a 
French Press. Centrifugatioll at 14,500 X g yielded a super-
natant that was further purified. The purification procedures 
included (NH4)2S04 fractionation, centrifugation at 105,000 X g, 
protamine sulfate precipitation of nueleic acids, hydroxyapatite 
adsorption, and DEAE-cellulo~e chromatography. A fraction 
from the chromatography procedure was diluted 1 :20 in 0.55 M 
potassium buffer, pH 7.3, 1 mM EDTA, 1 mM dithiothreitol, and 
20% glyceroL Aliquots of the diluted enzyme preparation were 
used for all assays unless indicated otherwise. The remainder of 
the pooled fractions was eoncelltrated and stored at -70°. 
The purification factor for the enzyme was 15.2-fold, and the 
preparation had a specific activity of 450 units per mg of protein 
(nanomoles of malonyl-CoA incorporated per min per mg) ,,,hen 
assayed with bovine serum albumin, and 265 units per mg with-
out bovine serum albumin. Higher specific activities (800 to 
1000 units per mg) were obserwd in fractions obtained from high 
pressure chromatography of the enzyme preparation (see below), 
but such fractions rapidly deteriorated to reach specific activities 
of 300 to 400 units per mg. 
jlfolewlar Weight-The molecular ,,-eight, estimated by chro-
matography! with Sepharo8e 4B (l'harmacia), was found to be 
2.5 and 3.0 X 106 g per mole ill b,·o trials. 
Products-The principal fatty aciel synthesized was palmitic 
acid.l Bovine serum albumin and th(, polysaccharides from Ji. 
phlei that stimulate fatty acid synthesis alter the relative per-
centages of fatty acids synthesized (13). 
S1tbstrates and Cofactors-The stimulatory effect of Loville 
serum albumin was indicated above' and has been investigated 
(13). Although the requirement for F:vIN was not absolute (62% 
activity when omitted), FAD was an ineffective substitute. 
1 This paper represents a summary of a more complete manu-
script that has been deposited 11S JBC Document No. 72M-1392. 
The deposited manuscript contains additional experimental dat.a 
and procedural details relevant to the conclusions advanced in 
the paper. Order for supplement.ary material should specify 
the title, author(s), and referenee to this paper and the JBC 
Document number, the form desired (microfiche or photocopy), 
and the number of copies desired. Orders should be addressed to' 
The Journal of Biological Chemistry, 9650 Rockville Pike, Be-
thesda, Maryland 20014 and must be accompanied by remittance 
to the order of the Journal in the amollnt of $2.50 per microfiche 
or $4.05 per photocopy of 27 pages. 
3517 
3518 
o 
N 
'Q 050M Phosphate 
E 
~ 
(f) 
(ii 
w 
:r: 
!-
z 
)-
<f) 
e 
u « 
r 
0-
l;;t 
u. 
12 
015 M Phosphate 
8 
010. M Phosphate 
10 20 40 50 60 
INCUBATION PERIOD (minutes) 
FIG. 1. The effect of various concentrations of phosphate on 
the fatty acid synthetase from Corynebacterium diphtheriae. En-
zyme assays were those described in the text except that the phos-
phate buffer concentrations were varied as indicated. 
Both DPNH ana TPNH were required and higher concentra-
+ 
tions of TPNH or the presence of DPN would not compensate 
for the omission of DPNH. The Km values for substrates were: 
DPNH, 2.5 J,tM; TP~H, 6 J,tM; acetyl-CoA, 25 J,tM; malonyl-CoA, 
10 J,tM. 
The elongation of a series of fatty acyl-CoA derivatives, in-
cluding propionic, isobutyric, and the even-numbered fatty acids 
with chain length::; between 4 and 16 were investigated. In all 
cases, activities were less than 10% of the vaille obtained with 
acetyl-CoA used as the substrate. 
Effect of Pho;;phate-A relatively high concentration of a multi-
valent anion was required for enzyme activity. Phosphate, at 
0.5 M, was the most effective of the anions tested, although citrate 
in lower concentrations was nearly equivalent to phosphate, and 
sulfate gave about 50% of the activity obtained with phosphate. 
The type of monovalent cation did not affect enzyme activity 
but Mg2+ caused precipitation in the assay mixture. The maxi-
mum activitv for each salt appeared to be more directly correlated 
with the con~entration of the anion, rather than the ionie strength 
of the assav medium. To determine whether the low enzyme 
activities observed for our enzyme in phosphatt' buffer of inter-
mediate concentrations (0.1 to 0.2 M) were due to the inactivation 
of the enzyme, reaction rates, with various concentrations of 
phosphate buffer, were determined (Fig. 1). In further experi-
ments, it was found that increasing the phosphate concentration 
(from 0.1 or 0.2 M to 0.5 M) at the midpoint of an incubation 
period inereased fatty acid synthesis. Since the enzyme was in 
a 0.5 M phosphate solution prior to being added to the assay mix-
ture, these results suggest that the catalytic efficiency of the 
TI I 
15 
N 
'0 
x 
E 
a. .., 
10 
>- ..s-
l- I() 
:; N 
i= <t 
~ 
I.tJ 
::E 
5 >-
N 
Z 
I.tJ 
20 10 
TIME [min) 
FIG. 2. High pressure chromatography of the fatty acid syn-
thetase preparation from Corynebacterium diphlheriae. .--., 
enzyme activity (disintegrations per min X 10-2); --, ultr~­
violet absorbance. Enzyme assays and chromatography condi-
tions are given in the text. 
synthetase is rapidly and reversibly affected by phosphate con-
centrations in the range of 0.1 to 0.5 M. 
When the enzyme was placed in 0.01 phosphate buffer at 37°, 
it was irreversibly inactivated within 5 min. At 0° the rate of 
inactivation \vas slower and was partially reversible. After 4 
hours in 0.01 M phosphate buffer, at 0°, the enzyme had retained 
10% of its original activity. When the phosphate concentration 
of this solution was raised to 0.6 M, enzyme activity increased, 
over a 20-hour period, to about 50% of that observed with an 
enzyme solution diluted to the same extent in 0.5 M phosphate 
and stored for 20 hours. A plot of log activity versus timel indi-
cated that the inactivation process involved more than one reac-
tion since there was a break between the linear portions of the 
plot. 
High Pressure Chromatography-Procedures for high pressure, 
gel permeation chromatography \vere developed by Dr. 1. Schec-
ter.l, 2 The stainless steel column (4 feet X % inch), packed 
with EM-Gel, type SI (Wolem) and the instrument equipped 
with a ultraviolet monitor was obtained from Chromatech, Inc. 
The column was treated with carbowax before used, and a 0.4 M 
potassium-phosphate buffer, pH 7.3 was the eluent. Conditions 
were: temperature, go; pressure, 300 p.s.i.; flow rate, 3 ml per 
min; fraction volume, 1.5 mL A synthetase preparation, specific 
activity about 400 units per mg, was applied to the column. 
As shown in Fig. 2, enzyme activity was present in one peak 
(1). Rechromatographyof Peak I yielded both peaks (J and II) 
with about equal areas, but rechromatography of Peak II gave 
onlv Peak II. These results suggest that the molecules of Peak I 
fra~tioll dissociate readily. To determine whether dissociation 
was involved in reversible inactivation of the enzyme in 0.01 M 
phosphate, enzyme was diluted into 0.01 M phosphate buffer at 
0°. After 4 hours, the phosphate concentration was adjusted to 
0.5 M in one-half of the solution and both samples were stored at 
2 Unpublished results, this laboratory. 
0° for 16 more hours. High pressure chromatography revealed 
that, compared to Fig. 2, Peak II was much larger than Peak I 
in both samples, but the ratios (1: II) of the peak areas (protein) 
for the inactivated (less than 10% of original activity) and reac-
tivated (40% of original activity) samples were not significantly 
differen LI 
Thus the lowering of phosphate concentration to 0.01 M resnlts 
in (a) a reversible inactivation without dissociation of the com-
plex, and (b) a dissociation that is irreversible under the condi-
tions used. These two processes seem to be distinct from the 
effects seen in the range of 0.1 to 0.5 M phosphate. 
DISCUSSION 
Comparing the properties of the C. diphtheriae, Ai. phlei, and 
yeast enzymes, it appears that the effect of phosphate concentra-
tion in the range of 0.1 to 0.5 M is distinctive for the C. diphtheriae 
synthetase. The enzyme from C. diphtheriae does resemble the 
Ai. phlei synthetase in that both lack stability in buffers of low 
iOllic strength (7) and that DPNH as well as TPNH are required 
electron donors (14). However, the Km values for TPNH and 
for malonyl-CoA are similar for the synthetascs from all three 
sources (5, 14, 15). The C. diphlheriae enzyme resembles the 
yeast enzyme with respect to molecular weight, products, Km for 
acetyl-eoA, and the response of the enzymes to bovine serum 
albumin and to the stimulatory polysaccharides from ilL. phlei 
(5, 7, 13), Thus a direct correlation of enzyme properties to 
supposed evolutionary development is not apparent when these 
three enzymes are considered; on the other hand, the presence of 
type I fatty acid synthetases in C. diphtheriae, ill. phlei, and 
3519 
Streptomyces coelicolor3 indicates that the multienzyme complex 
form may be common among the more advanced bacteria. 
Ackrwwledgmenl-The authors wish to thank Dr. Konrad 
Bloch for his advice and review throughout this study. 
HEFERENCES 
1. Hsu, H. Y., WASSON, G., AND PORTER, J. W. (1965) J. Bioi. 
Chern. 240, 3736 
2. BURTON, D. N., HAAVIK, A. G., AND PORTJi:U, J. W. (1968) 
Arch. Biochem. Biophys. 126, 141 
3. Hsu, R Y., AND YUN, S. L. (1970) Biochemis/)'Y 9,239 
4. SMITH, S., AND ABRAHAM, S. (1970) J. Biol. Chem. 245, 3209 
5. LYKJc;N, F. (1961) Fed. Proc. 20, 941 
6. HOLTJc;UMULL1'm, K. H., RIKGI{;LMANN. E., AND LYNEN, F. 
(1970) Hoppe.-Seyler's Z. PhY81:ol. Chern. 361, 1411 
7. BRINDLI,Y, D. N., MATSUMURA, S., AND BLOCH, K. (1969) 
Natltre 224, 666 
8. OVERATH, P., AND STUMPF, P. K. (1964) J. mol. Chern. 239, 
4103 
9. SrMoNI, R. D., CRIDDLE, R. S., AND STUMPF, P. K. (1967) J. 
Bioi. Chern. 242, 573 
lO. MAJICHUS, P. W., ALBERTS, A. W., AND VAm;LOs, P. R. (1964) 
Proc. Nal. Acad. Sci. U. S. A. 61,1231 
11. WAKIL, S. J., PUGH, E. L., AND SAUER, F. (1964) Proc. Nat. 
Acad. Sci. U. B. A. 52, 106 
12. DELO, J., ERNST-FoNBERG, M. L., AND BLOCH, K. (1971) Arch. 
Biochern. Biophys. 143, 384 
13. KNOCHE, H. W., ESDERS, T., KOTHS, K., AND BLOCH, K. (1973) 
J. Biol. Chern., 248, 2317 
14. Wmn;, H. B., III, MITSUHASHI, 0., AND BLOCH, IL (1971) 
J. Biol. Chern. 246, 4751 
15. lLTON, M., JEVANS, A. W .. McC"mTHY, E. D., VANCE, D., 
WHITE, H. B., III, AND BLOCH, K. (1971) Proc. Nat. Acad. 
Sci. U. S. A. 68, 87 
3 M. Weinrich, unpublished results, this laboratory. 


Characterization of a Fatty Acid Synthetase
from \u3ci\u3eCorynebacterium diphtheriae\u3c/i\u3e

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Characterization of a Fatty Acid Synthetase from \u3ci\u3eCorynebacterium diphtheriae\u3c/i\u3e

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