Efficient biotransformation of naringenin to naringenin α-glucoside, a novel α-glucosidase inhibitor, by amylosucrase from Deinococcus wulumuquiensis.

Amylosucrase (ASase) efficiently biosynthesizes α-glucoside using flavonoids as acceptor molecules and sucrose as a donor molecule. Here, ASase from Deinococcus wulumuqiensis (DwAS) biosynthesized more naringenin α-glucoside (NαG) with sucrose and naringenin as donor and acceptor molecules, respectively, than other ASases from Deinococcus sp. The biotransformation rate of DwAS to NαG was 21.3% compared to 7.1-16.2% for other ASases. Docking simulations showed that the active site of DwAS was more accessible to naringenin than those of others. The 217th valine in DwAS corresponded to the 221st isoleucine in Deinococcus geothermalis AS (DgAS), and the isoleucine possibly prevented naringenin from accessing the active site. The DwAS-V217I mutant had a significantly lower biosynthetic rate of NαG than DwAS. The kcat/Km value of DwAS with naringenin as the donor was significantly higher than that of DgAS and DwAS-V217I. In addition, NαG inhibited human intestinal α-glucosidase more efficiently than naringenin.

Spatial arrangement and density variations in the cell envelope of Deinococcus radiodurans.

The cell envelope of the poly-extremophile bacterium Deinococcus radiodurans is renowned for its highly organized structure and unique functional characteristics. In this bacterium, a precise regularity characterizes not just the S-layer, but it also extends to the underlying cell envelope layers, resulting in a dense and tightly arranged configuration. This regularity is attributed to a minimum of three protein complexes located at the outer membrane level. Together, they constitute a recurring structural unit that extends across the cell envelope, effectively tiling the entirety of the cell body. Nevertheless, a comprehensive grasp of the vacant spaces within each layer and their functional roles remains limited. In this study, we delve into these aspects by integrating the state of the art with structural calculations. This approach provides crucial evidence supporting an evolutive pressure intricately linked to surface phenomena depending on the environmental conditions.

The interconnecting hairpin extension "arm": An essential allosteric element of phytochrome activity.

In red-light sensing phytochromes, isomerization of the bilin chromophore triggers structural and dynamic changes across multiple domains, ultimately leading to control of the output module (OPM) activity. In between, a hairpin structure, "arm", extends from an interconnecting domain to the chromophore region. Here, by removing this protein segment in a bacteriophytochrome from Deinococcus radiodurans (DrBphP), we show that the arm is crucial for signal transduction. Crystallographic, spectroscopic, and biochemical data indicate that this variant maintains the properties of DrBphP in the resting state. Spectroscopic data also reveal that the armless systems maintain the ability to respond to light. However, there is no subsequent regulation of OPM activity without the arms. Thermal denaturation reveals that the arms stabilize the DrBphP structure. Our results underline the importance of the structurally flexible interconnecting hairpin extensions and describe their central role in the allosteric coupling of phytochromes.

Crystal structure of phosphate bound Acyl phosphatase mini-enzyme from Deinococcus radiodurans at 1Å resolution.

Acylphosphatase (Acp) is a hydrolase which specifically cleaves carboxyl-phosphate bond of intermediates of metabolic pathways. It is a small cytosolic enzyme found in both prokaryotic and eukaryotic organisms. Previous crystal structures of acylphosphatase from different organisms have provided insights into the active site but the complete understanding of substrate binding and catalytic mechanisms in acylphosphatase remain elusive. Here we report the crystal structure of phosphate bound acylphosphatase from a mesothermic bacterium, Deinococcus radiodurans (drAcp) at resolution of 1.0 Å. Our structural analysis shows how the terminal phosphate group of substrates is bound to the active site, highlighting the importance of arginine in substrate recognition, role of asparagine in mode of catalysis and shedding light on the reaction mechanism. Additionally, the protein can refold after thermal melting by gradually lowering the temperature. To further explore the dynamics of drAcp, molecular dynamics simulation of drAcp and homologs from thermophilic organisms were carried out which revealed similar root mean square fluctuation profile but drAcp showed comparatively higher fluctuations.

Interdigitated immunoglobulin arrays form the hyperstable surface layer of the extremophilic bacterium Deinococcus radiodurans.

Deinococcus radiodurans is an atypical diderm bacterium with a remarkable ability to tolerate various environmental stresses, due in part to its complex cell envelope encapsulated within a hyperstable surface layer (S-layer). Despite decades of research on this cell envelope, atomic structural details of the S-layer have remained obscure. In this study, we report the electron cryomicroscopy structure of the D. radiodurans S-layer, showing how it is formed by the Hexagonally Packed Intermediate-layer (HPI) protein arranged in a planar hexagonal lattice. The HPI protein forms an array of immunoglobulin-like folds within the S-layer, with each monomer extending into the adjacent hexamer, resulting in a highly interconnected, stable, sheet-like arrangement. Using electron cryotomography and subtomogram averaging from focused ion beam-milled D. radiodurans cells, we have obtained a structure of the cellular S-layer, showing how this HPI S-layer coats native membranes on the surface of cells. Our S-layer structure from the diderm bacterium D. radiodurans shows similarities to immunoglobulin-like domain-containing S-layers from monoderm bacteria and archaea, highlighting common features in cell surface organization across different domains of life, with connotations on the evolution of immunoglobulin-based molecular recognition systems in eukaryotes.

Controlled modulation of the dynamics of the Deinococcus grandis Dps N-terminal tails by divalent metals.

DNA-binding proteins from starved cells (Dps) are small multifunctional nanocages expressed by prokaryotes in acute oxidative stress conditions or during the starvation-induced stationary phase, as a bacterial defense mechanism. Dps proteins protect bacterial DNA from damage by either direct binding or by removing precursors of reactive oxygen species from solution. The DNA-binding properties of most Dps proteins studied so far are related to their unordered, flexible, N- and C-terminal extensions. In a previous work, we revealed that the N-terminal tails of Deinoccocus grandis Dps shift from an extended to a compact conformation depending on the ionic strength of the buffer and detected a novel high-spin ferrous iron center in the proximal ends of those tails. In this work, we further explore the conformational dynamics of the protein by probing the effect of divalent metals binding to the tail by comparing the metal-binding properties of the wild-type protein with a binding site-impaired D34A variant using size exclusion chromatography, dynamic light scattering, synchrotron radiation circular dichroism, and small-angle X-ray scattering. The N-terminal ferrous species was also characterized by Mössbauer spectroscopy. The results herein presented reveal that the conformation of the N-terminal tails is altered upon metal binding in a gradual, reversible, and specific manner. These observations may point towards the existence of a regulatory process for the DNA-binding properties of Dps proteins through metal binding to their N- and/or C-terminal extensions.

The SDBC is active in quenching oxidative conditions and bridges the cell envelope layers in Deinococcus radiodurans.

Deinococcus radiodurans is known for its remarkable ability to withstand harsh stressful conditions. The outermost layer of its cell envelope is a proteinaceous coat, the S-layer, essential for resistance to and interactions with the environment. The S-layer Deinoxanthin-binding complex (SDBC), one of the main units of the characteristic multilayered cell envelope of this bacterium, protects against environmental stressors and allows exchanges with the environment. So far, specific regions of this complex, the collar and the stalk, remained unassigned. Here, these regions are resolved by cryo-EM and locally refined. The resulting 3D map shows that the collar region of this multiprotein complex is a trimer of the protein DR_0644, a Cu-only superoxide dismutase (SOD) identified here to be efficient in quenching reactive oxygen species. The same data also showed that the stalk region consists of a coiled coil that extends into the cell envelope for ∼280 Å, reaching the inner membrane. Finally, the orientation and localization of the complex are defined by in situ cryo-electron crystallography. The structural organization of the SDBC couples fundamental UV antenna properties with the presence of a Cu-only SOD, showing here coexisting photoprotective and chemoprotective functions. These features suggests how the SDBC and similar protein complexes, might have played a primary role as evolutive templates for the origin of photoautotrophic processes by combining primary protective needs with more independent energetic strategies.

A multidomain connector links the outer membrane and cell wall in phylogenetically deep-branching bacteria.

Deinococcus radiodurans is a phylogenetically deep-branching extremophilic bacterium that is remarkably tolerant to numerous environmental stresses, including large doses of ultraviolet (UV) radiation and extreme temperatures. It can even survive in outer space for several years. This endurance of D. radiodurans has been partly ascribed to its atypical cell envelope comprising an inner membrane, a large periplasmic space with a thick peptidoglycan (PG) layer, and an outer membrane (OM) covered by a surface layer (S-layer). Despite intense research, molecular principles governing envelope organization and OM stabilization are unclear in D. radiodurans and related bacteria. Here, we report a electron cryomicroscopy (cryo-EM) structure of the abundant D. radiodurans OM protein SlpA, showing how its C-terminal segment forms homotrimers of 30-stranded ß-barrels in the OM, whereas its N-terminal segment forms long, homotrimeric coiled coils linking the OM to the PG layer via S-layer homology (SLH) domains. Furthermore, using protein structure prediction and sequence-based bioinformatic analysis, we show that SlpA-like putative OM-PG connector proteins are widespread in phylogenetically deep-branching Gram-negative bacteria. Finally, combining our atomic structures with fluorescence and electron microscopy of cell envelopes of wild-type and mutant bacterial strains, we report a model for the cell surface of D. radiodurans. Our results will have important implications for understanding the cell surface organization and hyperstability of D. radiodurans and related bacteria and the evolutionary transition between Gram-negative and Gram-positive bacteria.

The structural effect between the output module and chromophore-binding domain is a two-way street via the hairpin extension.

Signal transduction typically starts with either ligand binding or cofactor activation, eventually affecting biological activities in the cell. In red light-sensing phytochromes, isomerization of the bilin chromophore results in regulation of the activity of diverse output modules. During this process, several structural elements and chemical events influence signal propagation. In our study, we have studied the full-length bacteriophytochrome from Deinococcus radiodurans as well as a previously generated optogenetic tool where the native histidine kinase output module has been replaced with an adenylate cyclase. We show that the composition of the output module influences the stability of the hairpin extension. The hairpin, often referred as the PHY tongue, is one of the central structural elements for signal transduction. It extends from a distinct domain establishing close contacts with the chromophore binding site. If the coupling between these interactions is disrupted, the dynamic range of the enzymatic regulation is reduced. Our study highlights the complex conformational properties of the hairpin extension as a bidirectional link between the chromophore-binding site and the output module, as well as functional properties of diverse output modules.

Phosphorylation Regulation of a Histone-like HU Protein from Deinococcus radiodurans.

BACKGROUND: Histone-like proteins are small molecular weight DNA-binding proteins that are widely distributed in prokaryotes. These proteins have multiple functions in cellular structures and processes, including the morphological stability of the nucleoid, DNA compactness, DNA replication, and DNA repair. Deinococcus radiodurans, an extremophilic microorganism, has extraordinary DNA repair capability and encodes an essential histone-like protein, DrHU. OBJECTIVE: We aim to investigate the phosphorylation regulation role of a histone-like HU protein from Deinococcus radiodurans. METHODS: LC-MS/MS analysis was used to determine the phosphorylation site of endogenous DrHU. The predicted structure of DrHU-DNA was obtained from homology modeling (Swissmodel) using Staphylococcus aureus HU-DNA structure (PDB ID: 4QJU) as the starting model. Two types of mutant proteins T37E and T37A were generated to explore their DNA binding affinity. Complemented-knockout strategy was used to generate the ΔDrHU/pk-T37A and ΔDrHU/pk-T37E strains for growth curves and phenotypical analyses. RESULTS AND DISCUSSION: The phosphorylation site Thr37, which is present in most bacterial HU proteins, is located at the putative protein-DNA interaction interface of DrHU. Compared to the wild-type protein, one in which this threonine is replaced by glutamate to mimic a permanent state of phosphorylation (T37E) showed enhanced double-stranded DNA binding but a weakened protective effect against hydroxyl radical cleavage. Complementation of T37E in a DrHU-knockout strain caused growth defects and sensitized the cells to UV radiation and oxidative stress. CONCLUSIONS: Phosphorylation modulates the DNA-binding capabilities of the histone-like HU protein from D. radiodurans, which contributes to the environmental adaptation of this organism.

Conserved histidine and tyrosine determine spectral responses through the water network in Deinococcus radiodurans phytochrome.

Phytochromes are red light-sensing photoreceptor proteins that bind a bilin chromophore. Here, we investigate the role of a conserved histidine (H260) and tyrosine (Y263) in the chromophore-binding domain (CBD) of Deinococcus radiodurans phytochrome (DrBphP). Using crystallography, we show that in the H260A variant, the missing imidazole side chain leads to increased water content in the binding pocket. On the other hand, Y263F mutation reduces the water occupancy around the chromophore. Together, these changes in water coordination alter the protonation and spectroscopic properties of the biliverdin. These results pinpoint the importance of this conserved histidine and tyrosine, and the related water network, for the function and applications of phytochromes.

The structure of Deinococcus radiodurans transcriptional regulator HucR retold with the urate bound.

HucR is a MarR family protein of Deinococcus radiodurans, which binds tightly to the intergenic region of HucR and the uricase gene to inhibit their expression. Urate (or uric acid) antagonizes the repressor function of HucR by binding to HucR to impede its association with the cognate DNA. The previously reported crystal structure of HucR was without the bound urate showing significant structural homology to other MarR structures. In this paper, we report the crystal structure of HucR determined with the urate bound. However, despite the fact that the urate is found at a site well-known to harbor ligands in other MarR family proteins, the overall HucR structure indicates that no significant change in structure takes place with the urate bound. Structure analysis further suggests that the urate interaction in HucR is mediated by histidine/glutamate side chains and ordered water molecules stabilized by various residues. Such interaction is quite unique compared to other known structural interactions between urate and its binding proteins. Furthermore, structural comparison of the apo- and the urate bound forms allows us to hypothesize that the Trp20-mediated water network in the apo-form stabilizes the proper HucR fold for cognate DNA binding, and that urate binding, also via Trp20, and the consequent reorganization of water molecules in the binding pocket, likely disrupts the DNA binding configuration to result in the attenuated DNA binding.

The cryo-EM structure of the S-layer deinoxanthin-binding complex of Deinococcus radiodurans informs properties of its environmental interactions.

The radiation-resistant bacterium Deinococcus radiodurans is known as the world's toughest bacterium. The S-layer of D. radiodurans, consisting of several proteins on the surface of the cellular envelope and intimately associated with the outer membrane, has therefore been useful as a model for structural and functional studies. Its main proteinaceous unit, the S-layer deinoxanthin-binding complex (SDBC), is a hetero-oligomeric assembly known to contribute to the resistance against environmental stress and have porin functional features; however, its precise structure is unknown. Here, we resolved the structure of the SDBC at ∼2.5 Å resolution by cryo-EM and assigned the sequence of its main subunit, the protein DR_2577. This structure is characterized by a pore region, a massive ß-barrel organization, a stalk region consisting of a trimeric coiled coil, and a collar region at the base of the stalk. We show that each monomer binds three Cu ions and one Fe ion and retains one deinoxanthin molecule and two phosphoglycolipids, all exclusive to D. radiodurans. Finally, electrophysiological characterization of the SDBC shows that it exhibits transport properties with several amino acids. Taken together, these results highlight the SDBC as a robust structure displaying both protection and sieving functions that facilitates exchanges with the environment.

The hairpin extension controls solvent access to the chromophore binding pocket in a bacterial phytochrome: a UV-vis absorption spectroscopy study.

Solvent access to the protein interior plays an important role in the function of many proteins. Phytochromes contain a specific structural feature, a hairpin extension that appears to relay structural information from the chromophore to the rest of the protein. The extension interacts with amino acids near the chromophore, and hence shields the chromophore from the surrounding solvent. We envision that the detachment of the extension from the protein surface allows solvent exchange reactions in the vicinity of the chromophore. This can facilitate for example, proton transfer processes between solvent and the protein interior. To test this hypothesis, the kinetics of the protonation state of the biliverdin chromophore from Deinococcus radiodurans bacteriophytchrome, and thus, the pH of the surrounding solution, is determined. The observed absorbance changes are related to the solvent access of the chromophore binding pocket, gated by the hairpin extension. We therefore propose a model with an "open" (solvent-exposed, deprotonation-active on a (sub)second time-scale) state and a "closed" (solvent-gated, deprotonation inactive) state, where the hairpin fluctuates slowly between these conformations thereby controlling the deprotonation process of the chromophore on a minute time scale. When the connection between the hairpin and the biliverdin surroundings is destabilized by a point mutation, the amplitude of the deprotonation phase increases considerably. In the absence of the extension, the chromophore deprotonates essentially without any "gating". Hence, we introduce a straightforward method to study the stability and fluctuation of the phytochrome hairpin in its photostationary state. This approach can be extended to other chromophore-protein systems where absorption changes reflect dynamic processes of the protein.

Modular Assembly of Ordered Hydrophilic Proteins Improve Salinity Tolerance in Escherichia coli.

Most late embryogenesis abundant group 3 (G3LEA) proteins are highly hydrophilic and disordered, which can be transformed into ordered α-helices to play an important role in responding to diverse stresses in numerous organisms. Unlike most G3LEA proteins, DosH derived from Dinococcus radiodurans is a naturally ordered G3LEA protein, and previous studies have found that the N-terminal domain (position 1-103) of DosH protein is the key region for its folding into an ordered secondary structure. Synthetic biology provides the possibility for artificial assembling ordered G3LEA proteins or their analogues. In this report, we used the N-terminal domain of DosH protein as module A (named DS) and the hydrophilic domains (DrHD, BnHD, CeHD, and YlHD) of G3LEA protein from different sources as module B, and artificially assembled four non-natural hydrophilic proteins, named DS + DrHD, DS + BnHD, DS + CeHD, and DS + YlHD, respectively. Circular dichroism showed that the four hydrophile proteins were highly ordered proteins, in which the α-helix contents were DS + DrHD (56.1%), DS + BnHD (53.7%), DS + CeHD (49.1%), and DS + YLHD (64.6%), respectively. Phenotypic analysis showed that the survival rate of recombinant Escherichia coli containing ordered hydrophilic protein was more than 10% after 4 h treatment with 1.5 M NaCl, which was much higher than that of the control group. Meanwhile, in vivo enzyme activity results showed that they had higher activities of superoxide dismutase, catalase, lactate dehydrogenase and less malondialdehyde production. Based on these results, the N-terminal domain of DosH protein can be applied in synthetic biology due to the fact that it can change the order of hydrophilic domains, thus increasing stress resistance.

Functional and structural characterization of Deinococcus radiodurans R1 MazEF toxin-antitoxin system, Dr0416-Dr0417.

In prokaryotes, toxin-antitoxin (TA) systems are commonly found. They likely reflect the adaptation of pathogenic bacteria or extremophiles to various unfavorable environments by slowing their growth rate. Genomic analysis of the extremophile Deinococcus radiodurans R1 revealed the presence of eight type II TA systems, including the genes dr0417, dr0660, dr1530, dr0690, and dr1807. Expression of these toxin genes led to inhibition of Escherichia coli growth, whereas their antidote antitoxins were able to recover the growth defect. Remarkably, Dr0417 (DrMazF) showed endoribonuclease activity toward rRNAs as well as mRNAs, as determined by in vivo and in vitro RNA cleavage assays, and this activity was inhibited by Dr0416 (DrMazE). It was also found that the expression of dr0416-0417 module is directly regulated by the DrMazE-MazF complex. Furthermore, this TA module was induced under stress conditions and plays an important role in survival. To understand the regulatory mechanism at the molecular level, we determined the first high-resolution structures of DrMazF alone and of the DrMazE-MazF complex. In contrast with the hetero-hexameric state of typical MazE-MazF complexes found in other species, DrMazE-MazF crystal structure consists of a hetero-trimer, with the DNA-binding domain of DrMazE undergoing self-cleavage at the flexible linker loop. Our structure revealed that the unique residue R54 provides an additional positive charge to the substrate-binding pocket of DrMazF, its mutation significantly affects the endonuclease activity. Thus, our work reports the unique structural and biochemical features of the DrMazE-MazF system.

Signaling Mechanism of Phytochromes in Solution.

Phytochrome proteins guide the red/far-red photoresponse of plants, fungi, and bacteria. Crystal structures suggest that the mechanism of signal transduction from the chromophore to the output domains involves refolding of the so-called PHY tongue. It is currently not clear how the two other notable structural features of the phytochrome superfamily, the so-called helical spine and a knot in the peptide chain, are involved in photoconversion. Here, we present solution NMR data of the complete photosensory core module from Deinococcus radiodurans. Photoswitching between the resting and the active states induces changes in amide chemical shifts, residual dipolar couplings, and relaxation dynamics. All observables indicate a photoinduced structural change in the knot region and lower part of the helical spine. This implies that a conformational signal is transduced from the chromophore to the helical spine through the PAS and GAF domains. The discovered pathway underpins functional studies of plant phytochromes and may explain photosensing by phytochromes under biological conditions.

Deinococcus detaillensis sp. nov., isolated from humus soil in Antarctica.

A Gram-staining-positive, non-motile, coccus or short-rod-shaped bacterium, designated H1T, was isolated from a humus soil sample in the Detaille Island of Antarctica. The 16S rRNA gene sequence result indicated that strain H1T shared the highest 16S rRNA gene sequence identity with the type strain of Deinococcus alpinitundrae (96.2%). Growth of strain H1T occurred at 4-25 °C, pH 6.0-8.0 and in the presence of 0-1.0% NaCl (w/v). The respiratory quinone was MK-8. The major fatty acids were C16:0, C17:0 cyclo and summed feature 3 (C16:1 ω7c/C16:1 ω6c). The polar lipids were aminoglycophospholipid, aminophospholipid, glycolipid and glycophospholipid. The cell wall peptidoglycan type was A3ß. The genomic DNA G + C content was 61.3 mol%. The average nucleotide identity (ANI) between strain H1T and the closely related Deinococcus members was below the cut-off level (95-96%) for species identification. Based on the above results, strain H1T represents a novel species of the genus Deinococcus, for which the name Deinococcus detaillensis sp. nov. is proposed. Type strain is H1T (= CGMCC 1.13938T = JCM 33291T).

Crystal structure of the AhpD-like protein DR1765 from Deinococcus radiodurans R1.

Deinococcus radiodurans is well known for its extreme resistance to ionizing radiation (IR). Since reactive oxygen species generated by IR can damage various cellular components, D. radiodurans has developed effective antioxidant systems to cope with this oxidative stress. dr1765 from D. radiodurans is predicted to encode an alkyl hydroperoxidase-like protein (AhpD family), which is implicated in the reduction of hydrogen peroxide (H2O2) and organic hydroperoxides. In this study, we constructed a dr1765 mutant strain (Δdr1765) and examined the survival rate after H2O2 treatment. Δdr1765 showed a significant decrease in the H2O2 resistance compared to the wild-type strain. We also determined the crystal structure of DR1765 at 2.27 Å resolution. DR1765 adopted an all alpha helix protein fold representative of the AhpD-like superfamily. Structural comparisons of DR1765 with its structural homologues revealed that DR1765 possesses the Glu74-Cys86-Tyr88-Cys89-His93 signature motif, which is conserved in the proton relay system of AhpD. Complementation of Δdr1765 with dr1765 encoding C86A or C89A mutation failed to restore the survival rate to wild-type level. Taken together, these results suggest that DR1765 might function as an AhpD to protect cells from oxidative stress.

Bacterial Phytochrome as a Scaffold for Engineering of Receptor Tyrosine Kinases Controlled with Near-Infrared Light.

Optically controlled receptor tyrosine kinases (opto-RTKs) allow regulation of RTK signaling using light. Until recently, the majority of opto-RTKs were activated with blue-green light. Fusing a photosensory core module of Deinococcus radiodurans bacterial phytochrome (DrBphP-PCM) to the kinase domains of neurotrophin receptors resulted in opto-RTKs controlled with light above 650 nm. To expand this engineering approach to RTKs of other families, here we combined the DrBpP-PCM with the cytoplasmic domains of EGFR and FGFR1. The resultant Dr-EGFR and Dr-FGFR1 opto-RTKs are rapidly activated with near-infrared and inactivated with far-red light. The opto-RTKs efficiently trigger ERK1/2, PI3K/Akt, and PLCγ signaling. Absence of spectral crosstalk between the opto-RTKs and green fluorescent protein-based biosensors enables simultaneous Dr-FGFR1 activation and detection of calcium transients. Action mechanism of the DrBphP-PCM-based opto-RTKs is considered using the available RTK structures. DrBphP-PCM represents a versatile scaffold for engineering of opto-RTKs that are reversibly regulated with far-red and near-infrared light.