Isolation and characterization of the acetyl-CoA synthetase from Penicillium chrysogenum. Involvement of this enzyme in the biosynthesis of penicillins.

H Martínez-Blanco,A Reglero,M Fernández-Valverde,M.A Ferrero,M.A Moreno,M.A Peñalva,J.M Luengo

doi:10.1016/s0021-9258(18)42790-1

Abstract

Acetyl-CoA synthetase (ACS) of Penicillium chrysogenum was purified to homogeneity (745-fold) from fungal cultures grown in a chemically defined medium containing acetate as the main carbon source. The enzyme showed maximal rate of catalysis when incubated in 50 mM HCl-Tris buffer, pH 8.0, at 37 degrees C. Under these conditions, ACS showed hyperbolic behavior against acetate, CoA, and ATP; the Km values calculated for these substrates were 6.8, 0.18, and 17 mM, respectively. ACS recognized as substrates not only acetate but also several fatty acids ranging between C2 and C8 and some aromatic molecules (phenylacetic, 2-thiopheneacetic, and 3-thiopheneacetic acids). ATP can be replaced by ADP although, in this case, a lower activity was observed (37%). ACS in inhibited by some thiol reagents (5,5'-dithiobis(nitrobenzoic acid), N-ethylmaleimide, p-chloromercuribenzoate) and divalent cations (Zn2+, Cu2+, and Hg2+), whereas it was stimulated when the reaction mixtures contained 1 mM dithiothreitol, reduced glutathione, or 2-mercaptoethanol. The calculated molecular mass of ACS was 139 +/- 1 kDa, and the native enzyme is composed of two apparent identical subunits (70 kDa) in an alpha 2 oligomeric structure. ACS activity was regulated "in vivo" by carbon catabolite inactivation when glucose was taken up by cells in which the enzyme had been previously induced. This enzyme can be coupled "in vitro" to acyl-CoA:6-aminopenicillanic acid acyltransferase from P. chrysogenum, thus allowing the reconstitution of the functional enzymatic system which catalyzes the two latter reactions responsible for the biosynthesis of different penicillins. The ACS from Aspergillus nidulans can also be coupled to 6-aminopenicillanic acid acyltransferase to synthesize penicillins. These results strongly indicate that this enzyme can catalyze the activation (to their CoA thioesters) of some of the side-chain precursors required in these two fungi for the production of several penicillins. All these data are reported here for the first time.

Highlights

Acetyl-coA synthetase (ACS) of Penicillium chrys- The biosynthetic pathway of L-lysine and penicillins in ogenum was purified to homogeneity (746-fold) from Penicilliumchrysogenum andin Aspergillus nidulans is a fungal cultures grown ainchemically defined medium branchedroute which starts with the condensation of an containing acetateas the main carbon source
These results dures (15,16).we found a similar enzyme in a strain strongly indicate that this enzyme can catalyze the of Pseudomonas putidu (U) able to grow in a chemically activation of some of the side- defined medium containing phenylacetic acid (PAA) as the sole carbon source chain precursors required in these two fungi for the (17)
Time Course of the Appearance of Acetyl-coA Synthetase in P. chrysogenum-Acetyl-coA synthetase from P. chrysogenum (acetate:CoA ligase (AMP forming), EC 6.2.1.1) is an enzyme that catalyzes the activation of acetic acid to acetylCoA in thepresence of M%+,CoA, ATP, and acetate, according to thefollowing equation

Summary

INVOLVEMENT OF THIS ENZYME IN THE BIOSYNTHESIS OF PENICILLINS*

Acetyl-coA Synthetase Assay-ACS activity was evaluated by weight; about 10 mg of dry weight) were suspended in 25-ml Erlenmeasuring the rate of acetylhydroxamate formation in the presence meyer flasks containing 1.4 ml of0.06 M phosphate buffer, pH 6.5, of ATP, CoA, acetate, M e , and neutral hydroxylamine as has been and preincubated at 25 'C for 5 min in a thermostatically controlled described for other acyl-CoA activating enzymes (15-17). Samples of culture broth (10 were stopped by adding 450 pl of the ferric chloride reagent (see ml) were either filtered and lyophilized (for the determination of the below) and kept on ice for 10 min At this time the tubes were total @-lactamantibiotics, including 6-APA) or adjusted to pH 2.0, centrifuged in an Eppendorf 5414 microcentrifuge for 2 min and the extracted with isobutylacetate and the antibiotic-rich fraction (orred-purple color generated was measured a t 540 nm with a Shimadzu ganic phase) transferred to 50 mM phosphate buffer (l/lO, v/v) pH. The excess of KC1, which could have interfered insome experiments, was eliminated by filtration throughan Amicon membrane (exclusion size,

RESULTS AND DISCUSSION

Enzyme activity Specific activity

It can be observed that ACS recognizes better assubstrates

PAA plus acetate PAA plus acetate

Timr t h l