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

Abstract

<jats:p>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 <jats:italic>Ruegeria pomeroyi</jats:italic> and <jats:italic>Halomonas elongata</jats:italic>. 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, <jats:italic>N</jats:italic>-α-2 acetyl-<jats:sc>l</jats:sc>-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 <jats:sc>l</jats:sc>-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.</jats:p>

The ups and downs of ectoine: structural enzymology of a major microbial stress protectant and versatile nutrient

“…However, their occurrence in Archaea is restricted (Czech et al 2018a;Kunte et al 2014;Pastor et al 2010;Widderich et al 2016a) (Figure 2). Building on recent searches of the IMG/MER database of microbial genomes and metagenomic assemblies (Chen et al 2019), and considering only fully sequenced bacterial and archaeal genomes (Czech et al 2018a;Mais et al 2020), the distribution of the ectoine biosynthetic genes in 8 850 fully sequenced genomes (8557 Bacteria and 293 Archaea) was assessed ( Figure 2). In this dataset, 665 predicted ectoine producers originated from Bacteria while 11 representatives belong to the Archaea (Supplementary Figure S1).…”

“…These numbers are for obvious reasons only fleeting snapshots. Given that incompletely sequenced microbial genomes and metagenomic sequences were not taken into account, it is highly likely that the number of microbial ectoine producers will considerably exceed the number derived from the inspection of only fully sequenced microbial genomes (Mais et al 2020). Studies on the taxonomic affiliation of ectoine-synthesizing microorganisms revealed their presence in 10 bacterial and two archaeal phyla ( Figure 2).…”

“…While ectoine producers are often referred to in the literature as either halophilic or at least halotolerant bacteria, a closer analysis of the habitats of the 676 ectoine synthesizing microorganisms shows that 7% are associated with habitats exhibiting extreme conditions, 18% are associated with marine habitats, 27% are found in terrestrial habitats, 38% represent microbes associated with various types of hosts, and 8% are even found in freshwater ecosystems (Mais et al 2020). These numbers illustrate that microorganisms with rather different physiological attributes and growth characteristics can take advantage of ectoine synthesis to cope with environmental constrains (Supplementary Figure S1).…”

“…However, there are also many Archaea that use the salt-out response when they adjust to high salinity/ osmolarity environments and they synthesize a variety of compatible solutes, some of which also serve as excellent Figure 2: Phylogenetic distribution of ectoine-producing and ectoine-consuming microorganisms. The phylogenomic association of ectoine-producing and ectoineconsuming microorganisms was built on a recently reported data-set (Mais et al 2020). In this dataset, 665 bacterial and 11 archaeal ectoine-producing microorganisms, and 429 ectoine consuming bacteria are represented.…”

“…Ectoine producers were identified in the database search through the presence of the signature gene (ectC) for the ectoine synthase when juxtapositioned to other ectoine biosynthetic genes (ectAB) (Czech et al 2018a(Czech et al , 2019a. Ectoine consuming bacteria were identified through the presence of juxtapositioned genes (eutD/eutE) for the central catabolic enzyme bi-module, the ectoine hydrolase (EutD) and the DABA-deacetylase (EutE) (Mais et al 2020). For the studied 1105 microorganisms, 16S-rRNA data were collected and represented as collapsed clades.…”