Ono H, Sawada K, Khunajakr N, Tao T, Yamamoto M, Hiramoto M, Shinmyo A, Takano M, Murooka Y
Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamada-oka, Suita-shi, Osaka 565-0871, Japan.
J Bacteriol. 1999 Jan;181(1):91-9. doi: 10.1128/JB.181.1.91-99.1999.
1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic beta-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with L-glutamate. This enzyme required pyridoxal 5'-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25 degreesC and had Kms of 9.1 mM for L-glutamate and 4.5 mM for DL-ASA. DABA acetyltransferase catalyzed acetylation of DABA to gamma-N-acetyl-alpha,gamma-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20 degreesC in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15 degreesC in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0. 77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30 degreesC.
1,4,5,6-四氢-2-甲基-4-嘧啶羧酸（依克多因）是一种出色的渗透保护剂。通过对嗜盐栖热袍菌中参与依克多因生物合成途径的每种酶进行纯化和表征，阐明了从β-天冬氨酸半醛（ASA）合成依克多因的途径。2,4-二氨基丁酸（DABA）转氨酶可逆地催化该途径的第一步，即通过与L-谷氨酸转氨作用将ASA转化为DABA。该酶的活性和稳定性需要磷酸吡哆醛5'-磷酸和钾离子。凝胶过滤估计其表观分子量为260 kDa，而通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测得的分子量为44 kDa。该酶的最适pH为8.6，最适温度为25℃，对L-谷氨酸的Km值为9.1 mM，对DL-ASA的Km值为4.5 mM。DABA乙酰转移酶催化DABA与乙酰辅酶A乙酰化生成γ-N-乙酰-α,γ-二氨基丁酸（ADABA），在0.4 M NaCl存在下，其最适pH为8.2，最适温度为20℃。通过凝胶过滤测得其分子量为45 kDa。依克多因合酶催化ADABA环化生成依克多因，在0.5 M NaCl存在下，其最适pH为8.5至9.0，最适温度为15℃。通过SDS-PAGE测得该酶的表观分子量为19 kDa，在0.77 M NaCl存在下的Km值为8.4 mM。在低于30℃的温度下，NaCl（>2 M）和DABA（100 mM）可使DABA乙酰转移酶和依克多因合酶稳定。
