Physiological Role and Conformation of Ectoine
Shinichi NAGATA*a, Haruo MIMURAa, Kyoko ADACHIb,, Kenichi MOCHIDAb, Junichiro SETSUNEc, Erwin A. GALINSKId, Katsuhiro SUENOBUe, Masataka NAGAOKAe, and Tokio YAMABEf
a Kobe University of Mercantile Marine, 5-1-1 Fukae, Higashinada, Kobe 658, Japan
b Marine Biotechnology Institute, 1900 Sodeshicho, Shimizu 424, Japan
c Kobe University, 1-1 Rokkodai, Nada, Kobe 657, Japan
d Universitat Munster, Wilhelm-Klemm-Straβe 2, Munster 48149, Germany
e Institute for Fundamental Chemistry, 34-4 Takano-Nishihirakicho, Sakyo, Kyoto 606, Japan
f Kyoto University, Yoshidahonmachi, Sakyo, Kyoto 606, Japan
Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) is a cyclic amino acid and is well known as a compatible solute which is accumulated in halophilic and halotolerant bacteria to cope with the turgor pressure when grown in higher salinity. Role of ectoine in cell cytosol might be closely related to the protection from the key properties of macromolecular systems such as protein compartmentation and stability, enzymatic activity, and membrane fluidity which might be disrupted when salt concentrations reach high level. Halotolerant Brevibacterium sp. JCM 6894 is able to synthesize ectoine in the presence of >0.5 M NaC1, which leads to the ability to grow sufficiently at the higher osmolarity. Non-halophilic Escherichia coli can not synthesize ectoine in the cells and thus failed to grow at the higher salinity. Since E. coli possesses the Prop and ProU systems which are enable to take up ectoine, sufficient growth is permitted in the presence of >0.8 M NaC1 if ectoine is present extracellularly. Interestingly the accumulation of ectoine in the cells brought about the reduction of intracellular free Na+ and glutamate levels. To prove a reliable basis for investigations of structure/function correlations in ectoine, an attempt was tried to elucidate its stable conformation in terms of ab initio molecular orbital (MO) calculations and 1H-NMR spectroscopy at temperature ranges from-50 to 70℃. From the MO calculation of ectoine, the electronic structure of which was assumed to be a zwitterionic form, we obtained two stable structures, i.e., the COO group is in axial position and the other in equatorial position. The most stable structure of ectoine-water 1:1 complex was obtained for the hydrogen bonding interaction of one water molecule both at the oxygen atom of the COO group and at the hydrogen atom of the NH group. To probe whether there exists two conformations in ectoine or not we tried to measure the low-temperature NMR in CD3OD, but we could not detect the separate signals derived from them at-50℃. However, the triplet of H-4 eq shifted downfield with decline of temperature, from 3.920 at 70℃ to 3.967 ppm at -50℃. When the sample concentration increased 100 fold, from 0.5 to 50 mg/ml, the signal at H-4 eq shifted downfield by 0.012 ppm due to the molecular aggregation, which corresponds to the downfield shift by temperature lowering of ca. 30℃. By means of NOESY for ectoine we observed the signal via through-space interaction between H-4 ax and H-6 ax, which strongly indicated that ectoine is also existed in the structure with equatorial position of COO group. These experimental and theoretical data suggest that although we could not directly confirm the presence of two stable conformations for ectoine, there are little difference in energy between them. In relation to the structure of ectoine discussion will be extended for ectoine derivatives, e.g., hydroxyectoine and homoectoine.
