Characterization of biosynthetic enzymes for ectoine as a compatible solute in a moderately halophilic eubacterium, Halomonas elongata

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidine acid (ectoine) is an excellent osm oprotectant. The biosynthetic pathway of ectoine from aspartic beta-semiald ehyde (ASA), in Halomonas elongata, aas elucidated by purification and char acterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotrans ferase catalyzed reversively the first step of the pathway conversion of AS A to DABA by transamination with L-glutamate. This enzyme required pyridoxa l 5'-phosphate and potassium ions for its activity and stability. The gel f iltration estimated an apparent molecular mass of 260 kDa, whereas molecula r mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresi s (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25 degrees C and had K(m)s of 9.1 mM for L-glutamate and 4.5 mM for DL-ASA. DABA acetyltransferase catalyzed acetylation of DAB A to gamma-N-acetyl-alpha,gamma-diaminobutyric acid (ADABA) with acetyl coe nzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20 degrees C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ecto ine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15 degrees C in the presence of 0.5 M NaCl. This enzyme had an apparent mo lecular mass of 19 kDa by SDS-PAGE and a K-m 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 degr ees C.