Masai E, Shinohara S, Hara H, Nishikawa S, Katayama Y, Fukuda M
Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan.
J Bacteriol. 1999 Jan;181(1):55-62. doi: 10.1128/JB.181.1.55-62.1999.
Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds with guaiacyl moieties, which are converted into protocatechuate by the actions of lignin degradation enzymes in this strain. Protocatechuate is a key metabolite in the SYK-6 degradation of lignin compounds with guaiacyl moieties, and it is thought that it degrades to pyruvate and oxaloacetate via the protocatechuate 4,5-cleavage pathway. In a 10.5-kb EcoRI fragment carrying the protocatechuate 4,5-dioxygenase gene (ligAB) (Y. Noda, S. Nishikawa, K. Shiozuka, H. Kadokura, H. Nakajima, K. Yoda, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki. J. Bacteriol. 172:2704-2709, 1990), we found the ligI gene encoding 2-pyrone-4, 6-dicarboxylic acid (PDC) hydrolase. PDC hydrolase is a member of this pathway and catalyzes the interconversion between PDC and 4-carboxy-2-hydroxymuconic acid (CHM). The ligI gene is thought to be transcribed divergently from ligAB and consists of an 879-bp open reading frame encoding a polypeptide with a molecular mass of 32,737 Da. The ligI gene product (LigI), expressed in Escherichia coli, was purified to near-homogeneity and was estimated to be a monomer (31.6 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9. The optimum pH for hydrolysis of PDC is 8.5, the optimum pH for synthesis of PDC is 6.0 to 7.5, and the Km values for PDC and CHM are 74 and 49 microM, respectively. LigI activity was inhibited by the addition of thiol reagents, suggesting that the cysteine residue is a catalytic site. LigI is more resistant to metal ion inhibition than the PDC hydrolases of Pseudomonas ochraceae (K. Maruyama, J. Biochem. 93:557-565, 1983) and Comamonas testosteroni (P. J. Kersten, S. Dagley, J. W. Whittaker, D. M. Arciero, and J. D. Lipscomb, J. Bacteriol. 152:1154-1162, 1982). The insertional inactivation of the ligI gene in S. paucimobilis SYK-6 led to the complete loss of PDC hydrolase activity and to a growth defect on vanillic acid; it did not affect growth on syringic acid. These results indicate that the ligI gene is essential for the growth of SYK-6 on vanillic acid but is not responsible for the growth of SYK-6 on syringic acid.
少动鞘氨醇单胞菌SYK-6能够利用多种带有愈创木基部分的二聚木质素化合物生长，在该菌株中，这些化合物通过木质素降解酶的作用转化为原儿茶酸。原儿茶酸是SYK-6降解带有愈创木基部分的木质素化合物的关键代谢产物，据认为它通过原儿茶酸4,5-裂解途径降解为丙酮酸和草酰乙酸。在一个携带原儿茶酸4,5-双加氧酶基因（ligAB）的10.5 kb EcoRI片段中（Y. 野田、西川慎、塩冢健、门仓浩、中岛浩、与田洋、片山义、诸星直、原口彻、山崎明。《细菌学杂志》172:2704 - 2709, 1990），我们发现了编码2-吡喃酮-4,6-二羧酸（PDC）水解酶的ligI基因。PDC水解酶是该途径的一个成员，催化PDC和4-羧基-2-羟基粘康酸（CHM）之间的相互转化。据认为ligI基因与ligAB呈反向转录，由一个879 bp的开放阅读框组成，编码一个分子量为32,737 Da的多肽。在大肠杆菌中表达的ligI基因产物（LigI）被纯化至近乎均一，通过凝胶过滤色谱法估计为单体（31.6 kDa）。等电点测定为4.9。PDC水解的最适pH为8.5，PDC合成的最适pH为6.0至7.5，PDC和CHM的Km值分别为74和49 μM。添加硫醇试剂会抑制LigI活性，表明半胱氨酸残基是催化位点。与赭黄假单胞菌（K. 丸山，《生物化学杂志》93:557 - 565, 1983）和睾丸酮丛毛单胞菌（P. J. 克尔斯滕、S. 达格利、J. W. 惠特克、D. M. 阿奇罗、J. D. 利普斯科姆，《细菌学杂志》152:1154 - 1162, 1982）的PDC水解酶相比，LigI对金属离子抑制更具抗性。少动鞘氨醇单胞菌SYK-6中ligI基因的插入失活导致PDC水解酶活性完全丧失，并导致在香草酸上生长缺陷；但不影响在丁香酸上的生长。这些结果表明，ligI基因对于SYK-6在香草酸上的生长是必需的，但对SYK-6在丁香酸上的生长无作用。
