Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

Abstract

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-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°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°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 ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C 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°C.

Transcriptional regulation of ectoine catabolism in response to multiple metabolic and environmental cues

“…Apart from the high affinity of EnuR for N-(a)-ADABA, this compound has the additional advantage of being a specific ectoine-derived metabolite (Fig. 1), whereas DABA occurs also as an intermediate in other metabolic and biosynthetic processes in microorganisms (Ikai and Yamamoto, 1997;Du et al, 2013;Fidalgo et al, 2016), including the biosynthesis of ectoine (Ono et al, 1999) (Fig. 1).…”

“…The isomer of the high affinity EnuR ligand N-(a)-ADABA, N-(g)-ADABA, is an intermediate in ectoine biosynthesis ( Fig. 1) and serves as the main substrate for the ectoine synthase (EctC) (Ono et al, 1999;Widderich et al, 2016a). In some ectoine consumers (e.g., Halomonas elongata), N-(g)-ADABA can apparently also be generated through the ectoine hydrolase (EutD/DoeA) as a by-product of the main chemical reaction ( Fig.…”

“…These chemical reactions are transduced into a conformational change of the entire (Moran et al, 2004;Schulz et al, 2017). The sketches for the synthesis route for ectoine and hydroxyectoine (Ono et al, 1999;Bursy et al, 2007;St€ oveken et al, 2011) and that for their catabolism (Schwibbert et al, 2011;Schulz et al, 2017) are based on previously published data. All enzymes involved in ectoine and hydroxyectoine biosynthesis have been enzymatically studied (Ono et al, 1999;Bursy et al, 2007;St€ oveken et al, 2011), while EutD (DeoA) is the only enzyme from the catabolic route whose function has been experimentally assessed.…”

“…The sketches for the synthesis route for ectoine and hydroxyectoine (Ono et al, 1999;Bursy et al, 2007;St€ oveken et al, 2011) and that for their catabolism (Schwibbert et al, 2011;Schulz et al, 2017) are based on previously published data. All enzymes involved in ectoine and hydroxyectoine biosynthesis have been enzymatically studied (Ono et al, 1999;Bursy et al, 2007;St€ oveken et al, 2011), while EutD (DeoA) is the only enzyme from the catabolic route whose function has been experimentally assessed. The information on the generation of both N-(a)-ADABA and N-(g)-ADABA by the recombinant EutD (DoeA) enzyme from H. elongata is based on data reported by Schwibbert et al (Schwibbert et al, 2011). regulatory protein, which then dictates the DNA-binding and functional properties of MocR/GabR-type proteins to function either as activators or repressors (or both) of gene transcription (Belitsky and Sonenshein, 2002;Wiethaus et al, 2008;Edayathumangalam et al, 2013;Okuda et al, 2015a,b;Takenaka et al, 2015;Park et al, 2017;Tramonti et al, 2017;Wu et al, 2017).…”

Characterization of the recombinant diaminobutyric acid acetyltransferase from Methylophaga thalassica and Methylophaga alcalica

“…The recombinant EctA proteins from moderately halophilic M. thalassica and M. alcalica exist in vivo in the homodimeric form with subunit molecular masses of c. 20 kDa, temperature optima of 30 1C and a K m value for DABA of around 370 mM, and there were no requirements for divalent ions. The differences in the enzyme properties that correlated with the physiology of Methylophaga species were revealed and by comparison with DABAAT from the heterotroph H. elongata (Ono et al, 1999) and the his-tagged enzyme from methanotroph M. alcaliphilum (Reshetnikov et al, 2005) (Table 2).…”