Martínez-Blanco H, Reglero A, Fernández-Valverde M, Ferrero M A, Moreno M A, Peñalva M A, Luengo J M
Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de León, Spain.
J Biol Chem. 1992 Mar 15;267(8):5474-81.
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.
产黄青霉的乙酰辅酶A合成酶（ACS）通过在以醋酸盐作为主要碳源的化学限定培养基中培养的真菌培养物，被纯化至均一状态（745倍）。该酶在含有50 mM HCl - Tris缓冲液（pH 8.0），37℃孵育时表现出最大催化速率。在这些条件下，ACS对醋酸盐、辅酶A和ATP表现出双曲线行为；针对这些底物计算出的Km值分别为6.8、0.18和17 mM。ACS不仅识别醋酸盐为底物，还识别几种碳链长度在C2至C8之间的脂肪酸以及一些芳香分子（苯乙酸、2 - 噻吩乙酸和3 - 噻吩乙酸）。ATP可以被ADP替代，不过在这种情况下，观察到较低的活性（37%）。ACS受到一些硫醇试剂（5,5'-二硫代双（硝基苯甲酸）、N - 乙基马来酰亚胺、对氯汞苯甲酸）和二价阳离子（Zn2 +、Cu2 +和Hg2 +）的抑制，而当反应混合物含有1 mM二硫苏糖醇、还原型谷胱甘肽或2 - 巯基乙醇时，它会被激活。计算得出的ACS分子量为139±1 kDa，天然酶由两个明显相同的亚基（70 kDa）组成α2寡聚结构。当葡萄糖被先前已诱导该酶的细胞摄取时，ACS活性在“体内”受到碳分解代谢失活的调节。这种酶可以在“体外”与产黄青霉的酰基辅酶A：6 - 氨基青霉烷酸酰基转移酶偶联，从而实现功能性酶系统的重构，该系统催化负责不同青霉素生物合成的后两个反应。来自构巢曲霉的ACS也可以与6 - 氨基青霉烷酸酰基转移酶偶联以合成青霉素。这些结果有力地表明，这种酶可以催化这两种真菌生产几种青霉素所需的一些侧链前体的激活（形成它们的辅酶A硫酯）。所有这些数据首次在此报道。
