Pelmont J, Barrelle M, Hauteville M, Gamba D, Romdhane M, Dardas A, Beguin C
Biochimie. 1985 Sep;67(9):973-86. doi: 10.1016/s0300-9084(85)80292-3.
A lignin model compound, named in short guaiagylglycerol beta-guaiacyl ether (GGE), contains the beta-0-4 ether linkage that is common in the chemical structure of lignin. A Pseudomonas sp. (GU5) had been isolated as an organism able to grow with GGE as the sole source of carbon and energy. When grown on vanillate, the bacteria contained a NAD+ -dependent dehydrogenase converting GGE to a 355 nm absorbing product. The enzyme, named GGE-dehydrogenase, was purified about 160-fold using gel permeation, ion exchange on DEAE-Sephadex, and dye-ligand affinity chromatography. The new protein was about 52 kDa in apparent size with but one polypeptide chain after denaturation and reduction. According to several criteria, the product of GGE oxidation (Km = 12 microM) was identified as the corresponding conjugated ketone at the alpha-carbon of the C3 side-chain. The secondary alcohol function in GGE was apparently the sole target of the enzyme action. However the conversion of GGE into ketone catalyzed by the enzyme was only partial, and did not exceed 50%, probably because only one of the alpha-enantiomers was susceptible to enzyme attack. In contrast the ketone, either made by organic synthesis or by enzymic oxidation of GGE, could be totally reduced back to GGE (Km = 13 microM at pH 8.4, 8 microM at neutral pH), with NADH as the reductant, as confirmed by UV absorption and NMR spectra. Other model compounds with no primary alcoholic function, ether linkage or phenolic group were also substrates for the enzyme, confirming the specificity of GGE-dehydrogenase for the alpha-carbon position. Conjugation of the alpha-ketone with an adjacent phenolic nucleus interfered strongly with equilibrium constants and redox potentials of the system according to pH, and the enzyme displayed widely different optima with pH over 9 when oxidizing GGE, below 7 when reducing the ketone. Equilibrium studies showed that the ketone/GGE potential was -0.37 volt at pH 8.7, -0.23 volt at pH 7 (30 degrees C). The significance of this new dehydrogenase and its properties are discussed, especially in the general concern of lignin biodegradation.
一种木质素模型化合物，简称为愈创木基甘油-β-愈创木基醚（GGE），含有木质素化学结构中常见的β-O-4醚键。一种假单胞菌属菌株（GU5）已被分离出来，它能够以GGE作为唯一的碳源和能源生长。当在香草酸盐上生长时，该细菌含有一种依赖NAD⁺的脱氢酶，可将GGE转化为一种在355nm处有吸收的产物。这种酶被命名为GGE脱氢酶，通过凝胶渗透、DEAE-葡聚糖离子交换和染料配体亲和色谱法进行了约160倍的纯化。这种新蛋白质的表观大小约为52kDa，变性和还原后只有一条多肽链。根据多项标准，GGE氧化产物（Km = 12μM）被鉴定为C3侧链α-碳上的相应共轭酮。GGE中的仲醇功能显然是酶作用的唯一靶点。然而，该酶催化GGE转化为酮的反应只是部分反应，不超过50%，可能是因为只有一种α-对映体易受酶的攻击。相比之下，无论是通过有机合成还是通过GGE的酶促氧化制备的酮，都可以以NADH作为还原剂完全还原回GGE（在pH 8.4时Km = 13μM，在中性pH时Km = 8μM），这通过紫外吸收和核磁共振光谱得到了证实。其他没有伯醇功能、醚键或酚羟基的模型化合物也是该酶的底物，这证实了GGE脱氢酶对α-碳位置的特异性。α-酮与相邻酚核的共轭根据pH强烈干扰了该系统的平衡常数和氧化还原电位，并且该酶在氧化GGE时在pH超过9时有广泛不同的最适值，在还原酮时在pH低于7时有广泛不同的最适值。平衡研究表明，在pH 8.7时酮/GGE电位为-0.37伏，在pH 7时（30℃）为-0.23伏。讨论了这种新脱氢酶的意义及其性质，特别是在木质素生物降解的总体研究中。
