Degradation of the microbial stress protectants and chemical chaperones ectoine and hydroxyectoine by a bacterial hydrolase-deacetylase complex.

When faced with increased osmolarity in the environment, many bacterial cells accumulate the compatible solute ectoine and its derivative 5-hydroxyectoine. Both compounds are not only potent osmostress protectants, but also serve as effective chemical chaperones stabilizing protein functionality. Ectoines are energy-rich nitrogen and carbon sources that have an ecological impact that shapes microbial communities. Although the biochemistry of ectoine and 5-hydroxyectoine biosynthesis is well understood, our understanding of their catabolism is only rudimentary. Here, we combined biochemical and structural approaches to unravel the core of ectoine and 5-hydroxy-ectoine catabolisms. We show that a conserved enzyme bimodule consisting of the EutD ectoine/5-hydroxyectoine hydrolase and the EutE deacetylase degrades both ectoines. We determined the high-resolution crystal structures of both enzymes, derived from the salt-tolerant bacteria Ruegeria pomeroyi and Halomonas elongata These structures, either in their apo-forms or in forms capturing substrates or intermediates, provided detailed insights into the catalytic cores of the EutD and EutE enzymes. The combined biochemical and structural results indicate that the EutD homodimer opens the pyrimidine ring of ectoine through an unusual covalent intermediate, N-α-2 acetyl-l-2,4-diaminobutyrate (α-ADABA). We found that α-ADABA is then deacetylated by the zinc-dependent EutE monomer into diaminobutyric acid (DABA), which is further catabolized to l-aspartate. We observed that the EutD-EutE bimodule synthesizes exclusively the α-, but not the γ-isomers of ADABA or hydroxy-ADABA. Of note, α-ADABA is known to induce the MocR/GabR-type repressor EnuR, which controls the expression of many ectoine catabolic genes clusters. We conclude that hydroxy-α-ADABA might serve a similar function.

The architecture of the diaminobutyrate acetyltransferase active site provides mechanistic insight into the biosynthesis of the chemical chaperone ectoine.

Ectoine is a solute compatible with the physiologies of both prokaryotic and eukaryotic cells and is widely synthesized by bacteria as an osmotic stress protectant. Because it preserves functional attributes of proteins and macromolecular complexes, it is considered a chemical chaperone and has found numerous practical applications. However, the mechanism of its biosynthesis is incompletely understood. The second step in ectoine biosynthesis is catalyzed by l-2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), which transfers the acetyl group from acetyl-CoA to EctB-formed l-2,4-diaminobutyrate (DAB), yielding N-γ-acetyl-l-2,4-diaminobutyrate (N-γ-ADABA), the substrate of ectoine synthase (EctC). Here, we report the biochemical and structural characterization of the EctA enzyme from the thermotolerant bacterium Paenibacillus lautus (Pl). We found that (Pl)EctA forms a homodimer whose enzyme activity is highly regiospecific by producing N-γ-ADABA but not the ectoine catabolic intermediate N-α-acetyl-l-2,4-diaminobutyric acid. High-resolution crystal structures of (Pl)EctA (at 1.2-2.2 Å resolution) (i) for its apo-form, (ii) in complex with CoA, (iii) in complex with DAB, (iv) in complex with both CoA and DAB, and (v) in the presence of the product N-γ-ADABA were obtained. To pinpoint residues involved in DAB binding, we probed the structure-function relationship of (Pl)EctA by site-directed mutagenesis. Phylogenomics shows that EctA-type proteins from both Bacteria and Archaea are evolutionarily highly conserved, including catalytically important residues. Collectively, our biochemical and structural findings yielded detailed insights into the catalytic core of the EctA enzyme that laid the foundation for unraveling its reaction mechanism.

Biosynthesis of the Stress-Protectant and Chemical Chaperon Ectoine: Biochemistry of the Transaminase EctB.

Bacteria frequently adapt to high osmolarity surroundings through the accumulation of compatible solutes. Ectoine is a prominent member of these types of stress protectants and is produced via an evolutionarily conserved biosynthetic pathway beginning with the L-2,4-diaminobutyrate (DAB) transaminase (TA) EctB. Here, we studied EctB from the thermo-tolerant Gram-positive bacterium Paenibacillus lautus (Pl) and show that this tetrameric enzyme is highly tolerant to salt, pH, and temperature. During ectoine biosynthesis, EctB converts L-glutamate and L-aspartate-beta-semialdehyde into 2-oxoglutarate and DAB, but it also catalyzes the reverse reaction. Our analysis unravels that EctB enzymes are mechanistically identical to the PLP-dependent gamma-aminobutyrate TAs (GABA-TAs) and only differ with respect to substrate binding. Inspection of the genomic context of the ectB gene in P. lautus identifies an unusual arrangement of juxtapositioned genes for ectoine biosynthesis and import via an Ehu-type binding-protein-dependent ABC transporter. This operon-like structure suggests the operation of a highly coordinated system for ectoine synthesis and import to maintain physiologically adequate cellular ectoine pools under osmotic stress conditions in a resource-efficient manner. Taken together, our study provides an in-depth mechanistic and physiological description of EctB, the first enzyme of the ectoine biosynthetic pathway.

Role of the Extremolytes Ectoine and Hydroxyectoine as Stress Protectants and Nutrients: Genetics, Phylogenomics, Biochemistry, and Structural Analysis.

Fluctuations in environmental osmolarity are ubiquitous stress factors in many natural habitats of microorganisms, as they inevitably trigger osmotically instigated fluxes of water across the semi-permeable cytoplasmic membrane. Under hyperosmotic conditions, many microorganisms fend off the detrimental effects of water efflux and the ensuing dehydration of the cytoplasm and drop in turgor through the accumulation of a restricted class of organic osmolytes, the compatible solutes. Ectoine and its derivative 5-hydroxyectoine are prominent members of these compounds and are synthesized widely by members of the Bacteria and a few Archaea and Eukarya in response to high salinity/osmolarity and/or growth temperature extremes. Ectoines have excellent function-preserving properties, attributes that have led to their description as chemical chaperones and fostered the development of an industrial-scale biotechnological production process for their exploitation in biotechnology, skin care, and medicine. We review, here, the current knowledge on the biochemistry of the ectoine/hydroxyectoine biosynthetic enzymes and the available crystal structures of some of them, explore the genetics of the underlying biosynthetic genes and their transcriptional regulation, and present an extensive phylogenomic analysis of the ectoine/hydroxyectoine biosynthetic genes. In addition, we address the biochemistry, phylogenomics, and genetic regulation for the alternative use of ectoines as nutrients.