Kauppinen S, Christgau S, Kofod L V, Halkier T, Dörreich K, Dalbøge H
GeneSearch, Novo Nordisk A/S, Novo Allé, Bagsvaerd, Denmark.
J Biol Chem. 1995 Nov 10;270(45):27172-8. doi: 10.1074/jbc.270.45.27172.
A rhamnogalacturonan acetylesterase (RGAE) was purified to homogeneity from the filamentous fungus Aspergillus aculeatus, and the NH2-terminal amino acid sequence was determined. Full-length cDNAs encoding the enzyme were isolated from an A. aculeatus cDNA library using a polymerase chain reaction-generated product as a probe. The 936-base pair rha1 cDNA encodes a 250-residue precursor protein of 26,350 Da, including a 17-amino acid signal peptide. The rha1 cDNA was overexpressed in Aspergillus oryzae, a filamentous fungus that does not possess RGAE activity, and the recombinant enzyme was purified and characterized. Mass spectrometry of the native and recombinant RGAE revealed that the enzymes are heterogeneously glycosylated. In addition, the observed differences in their molecular masses, lectin binding patterns, and monosaccharide compositions indicate that the glycan moieties on the two enzymes are structurally different. The RGAE was shown to act in synergy with rhamnogalacturonase A as well as rhamnogalacturonase B from A. aculeatus in the degradation of apple pectin rhamnogalacturonan. RNA gel blot analyses indicate that the expression of rhamnogalacturonan degrading enzymes by A. acculeatus is regulated at the level of transcription and is subjected to carbon catabolite repression by glucose.
从丝状真菌棘孢曲霉中纯化出一种鼠李半乳糖醛酸乙酰酯酶（RGAE）并使其达到同质，同时测定了其氨基末端氨基酸序列。以聚合酶链反应生成的产物为探针，从棘孢曲霉cDNA文库中分离出编码该酶的全长cDNA。936个碱基对的rha1 cDNA编码一个由250个氨基酸残基组成、分子量为26,350 Da的前体蛋白，其中包括一个17个氨基酸的信号肽。rha1 cDNA在不具有RGAE活性的丝状真菌米曲霉中过表达，对重组酶进行了纯化和表征。对天然和重组RGAE进行质谱分析表明，这些酶存在异质性糖基化。此外，观察到的它们分子量、凝集素结合模式和单糖组成的差异表明，这两种酶上的聚糖部分在结构上是不同的。结果表明，RGAE在降解苹果果胶鼠李半乳糖醛酸聚糖时，与来自棘孢曲霉的鼠李半乳糖醛酸酶A以及鼠李半乳糖醛酸酶B协同作用。RNA凝胶印迹分析表明，棘孢曲霉对鼠李半乳糖醛酸降解酶的表达在转录水平受到调控，并受到葡萄糖的碳分解代谢物阻遏。
