Abstract
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.
Highlights
Fluctuations in the environmental osmolarity are frequently encountered challenges that most freeliving microorganisms have to cope with in their ecophysiologically varied habitats (Galinski and Trüper, 1994; Kempf and Bremer, 1998; Wood et al, 2001; Gunde-Cimerman et al, 2018)
multi-angle light scattering (MALS) coupled to refractive index (RI) experiments unambiguously showed that the protein elutes from the size-exclusion chromatography (SEC) column with an absolute molecular mass of approximately 200 kDa, suggesting that (Pl)EctB forms a homotetramer in solution (Figure 2B)
Given the interest in TAs for practical purposes (Steffen-Munsberg et al, 2015), the robust P. lautus EctB protein might provide opportunities to exploit this enzyme for biotechnological applications. (Pl)EctB differs from its H. elongata counterpart (Ono et al, 1999), as it does not need higher concentrations of K+ for its enzyme activity or stability (Figure 3C)
Summary
Fluctuations in the environmental osmolarity are frequently encountered challenges that most freeliving microorganisms have to cope with in their ecophysiologically varied habitats (Galinski and Trüper, 1994; Kempf and Bremer, 1998; Wood et al, 2001; Gunde-Cimerman et al, 2018). When the external osmolarity suddenly drops, increased water influx can affect cellular integrity as the result of an excessive increase in turgor (Booth, 2014; Cox et al, 2018). To avert these detrimental effects, microorganisms actively modulate the osmotic potential of their crowded cytoplasm in order to indirectly guide and scale compensatory water fluxes (Wood, 2011; van den Berg et al, 2017; Bremer and Krämer, 2019)
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