Salt Mediated Modulation of Autolysis of Thermolysin-Like Proteinase, Salilysin, Isolated from a Moderate Halophile, Chromohalobacter salexigens DSM3043.

Halophilic salilysin is first synthesized as a pro-form, which has been shown autolysis activity to process pro-region (55 amino acids long) three times to form intermediate 1 (I1), intermediate 2 (I2) and final mature (M) salilysin. The autolysis of I1- to M-form salilysin in vitro was significantly accelerated with increasing NaCl concentration up to 4 M. Strong salting-out salts, (NH4)2SO4, Na2SO4 and MgSO4, were more effective, suggesting that autolysis is enhanced by inter-molecular association or structure compaction or both. However, MgCl2, a salting-in salt, was also effective, suggesting that other mechanisms, such as charge shielding and ionic binding to this halophilic protein, operated. Autolytic cleavage at site 3 resulted in mixed formation of correctly and incorrectly processed mature forms in the absence of salt, indicating that salt affected the accuracy of autolytic cleavage reaction. Far UV circular dichroism (CD) measurements indicated that E167A pro-salilysin showed an identical CD spectrum to the wild-type mature salilysin, suggesting pro-form has a proper fold for proteolytic activity. Thermal scanning indicated that E167A pro-salilysin was more heat-stable by ~ 10 °C than mature form. The CD spectra, thermal stability and modeling structure of salilysin clearly suggested that pro-salilysin is folded to the same structure as native form and is functional for autolysis.

Salt-enhanced processing, proteolytic activity and stability of halophilic thermolysin-like proteinase, salilysin, isolated from a moderate halophile, Chromohalobacter salexigens DSM3043.

Moderately halophilic bacterium, Chromohalobacter salexigens DSM3043, has a gene Csal_2537 encoding thermolysin-like M4 proteinase. This gene was cloned to pET expression vectors, resulting in high expression of recombinant proteinase, named as salilysin (salinity-dependent thermolysin-like proteinase), in Escherichia coli cytoplasm. This gene encodes precursor form of salilysin containing 348 amino acid residues (Pro-salilysin) consisting of 55 amino acids pro-sequence and following mature proteinase. Pro-sequence was cleaved three times to form intermediate 1, intermediate 2 and final mature salilysin. The processing rate was greatly accelerated in a salt concentration-dependent manner. Purified inactive mutant Pro-E167A-salilysin was correctly processed by purified mature salilysin, indicating that autolysis and inter-molecular processing occurred in its maturation processes. Proteolytic activity of mature salilysin against both peptide and protein substrates was also enhanced along with the addition of higher concentration of salt, 0-3.2 M NaCl, consistent with its halophilic origin. Mature salilysin was stabilized by ~8 °C in the presence of 1 M NaCl by thermal scanning using circular dichroism. One of the precursor form, intermediate 1, showed ~20 °C higher denaturation temperature than mature form, suggesting rigid and stable structure of this precursor form.

The crystal structure of the tetrameric DABA-aminotransferase EctB, a rate-limiting enzyme in the ectoine biosynthesis pathway.

l-2,4-diaminobutyric acid (DABA) aminotransferases can catalyze the formation of amines at the distal ω-position of substrates, and is the intial and rate-limiting enzyme in the biosynthesis pathway of the cytoprotecting molecule (S)-2-methyl-1,4,5,6-tetrahydro-4-pyrimidine carboxylic acid (ectoine). Although there is an industrial interest in the biosynthesis of ectoine, the DABA aminotransferases remain poorly characterized. Herein, we present the crystal structure of EctB (2.45 Å), a DABA aminotransferase from Chromohalobacter salexigens DSM 3043, a well-studied organism with respect to osmoadaptation by ectoine biosynthesis. We investigate the enzyme's oligomeric state to show that EctB from C. salexigens is a tetramer of two functional dimers, and suggest conserved recognition sites for dimerization that also includes the characteristic gating loop that helps shape the active site of the neighboring monomer. Although ω-transaminases are known to have two binding pockets to accommodate for their dual substrate specificity, we herein provide the first description of two binding pockets in the active site that may account for the catalytic character of DABA aminotransferases. Furthermore, our biochemical data reveal that the EctB enzyme from C. salexigens is a thermostable, halotolerant enzyme with a broad pH tolerance which may be linked to its tetrameric state. Put together, this study creates a solid foundation for a deeper structural understanding of DABA aminotransferases and opening up for future downstream studies of EctB's catalytic character and its redesign as a better catalyst for ectoine biosynthesis. In summary, we believe that the EctB enzyme from C. salexigens can serve as a benchmark enzyme for characterization of DABA aminotransferases. DATABASE: Structural data are available in PDB database under the accession number 6RL5.

Purification and characterization of halophilic lipase of Chromohalobacter sp. from ancient salt well.

A halophilic lipase (LipS2) was produced by Chromohalobacter canadensis strain which was isolated from ancient salt well of Zigong, China. LipS2 was purified to homogeneity and showed a single band with molecular mass of 58 kDa by SDS-PAGE. LipS2 preferred middle-to-long acyl chain esters with C14 triglycerides as optimum substrate. It was noteworthy that LipS2 displayed efficient hydrolysis activity to some vegetable oils which were composed of polyunsaturated fatty acid. LipS2 showed high activity in range of 2.5-3.5 M NaCl, no activity without salt. Optimum temperature and pH were 55 °C and pH 8.5, respectively. Notably, the thermostability and pH stability of LipS2, varying with salt concentration, reached optimum in the presence of 3.0 M NaCl. LipS2 was stimulated by Ca2+ and Mg2+ , inhibited by Zn2+ , Cu2+ , Mn2+ , Fe2+ , and Hg2+ . Moreover, LipS2 displayed significant tolerance to organic solvents including methanol, ethanol, ethyl acetate and acetone, especially, LipS2 activity was enhanced markedly by the hexane and benzene. Non-ionic surfactants increased LipS2 activity, while ionic surfactants decreased activity. This was the first report on halophilic lipase of Chromohalobacter from ancient salt well. The results suggested that LipS2 may have considerable potential for biotechnological applications.

Glycine Betaine Monooxygenase, an Unusual Rieske-Type Oxygenase System, Catalyzes the Oxidative N-Demethylation of Glycine Betaine in Chromohalobacter salexigens DSM 3043.

Although some bacteria, including Chromohalobacter salexigens DSM 3043, can use glycine betaine (GB) as a sole source of carbon and energy, little information is available about the genes and their encoded proteins involved in the initial step of the GB degradation pathway. In the present study, the results of conserved domain analysis, construction of in-frame deletion mutants, and an in vivo functional complementation assay suggested that the open reading frames Csal_1004 and Csal_1005, designated bmoA and bmoB, respectively, may act as the terminal oxygenase and the ferredoxin reductase genes in a novel Rieske-type oxygenase system to convert GB to dimethylglycine in C. salexigens DSM 3043. To further verify their function, BmoA and BmoB were heterologously overexpressed in Escherichia coli, and 13C nuclear magnetic resonance analysis revealed that dimethylglycine was accumulated in E. coli BL21(DE3) expressing BmoAB or BmoA. In addition, His-tagged BmoA and BmoB were individually purified to electrophoretic homogeneity and estimated to be a homotrimer and a monomer, respectively. In vitro biochemical analysis indicated that BmoB is an NADH-dependent flavin reductase with one noncovalently bound flavin adenine dinucleotide (FAD) as its prosthetic group. In the presence of BmoB, NADH, and flavin, BmoA could aerobically degrade GB to dimethylglycine with the concomitant production of formaldehyde. BmoA exhibited strict substrate specificity for GB, and its demethylation activity was stimulated by Fe2+ Phylogenetic analysis showed that BmoA belongs to group V of the Rieske nonheme iron oxygenase (RO) family, and all the members in this group were able to use quaternary ammonium compounds as substrates.IMPORTANCE GB is widely distributed in nature. In addition to being accumulated intracellularly as a compatible solute to deal with osmotic stress, it can be utilized by many bacteria as a source of carbon and energy. However, very limited knowledge is presently available about the molecular and biochemical mechanisms for the initial step of the aerobic GB degradation pathway in bacteria. Here, we report the molecular and biochemical characterization of a novel two-component Rieske-type monooxygenase system, GB monooxygenase (BMO), which is responsible for oxidative demethylation of GB to dimethylglycine in C. salexigens DSM 3043. The results gained in this study extend our knowledge on the catalytic reaction of microbial GB degradation to dimethylglycine.

Proteolytic activity of recombinant DegP from Chromohalobacter salexigens BKL5

Background: DegP is a serine protease that specifically cleaves and refolds unfolding proteins in the periplasmic space of the cells. To date, there is no information regarding DegP from halophilic bacteria. Chromohalobacter salexigens BKL5 is a moderately halophilic bacterium that has the ability to grow in a media containing more than 15% salt. Therefore, the objectives of this work were to clone and overexpress DegP-encoding gene from C. salexigens BKL5 and characterize its biochemical properties. Results: DegP-encoding gene was overexpressed in Escherichia coli BL21(DE3) CodonPlus in an active form. SDS-PAGE analysis showed that the molecular weight of the recombinant DegP was 45 kDa. Size-exclusion chromatography analysis suggested that recombinant DegP was present in two multimeric states, hexameric and dodecameric, with molecular weights of 297.9 and 579.12 kDa, respectively. Both conformations were enzymatically active when casein was used as substrate for enzymatic assay. Circular dichroism analysis showed that recombinant DegP was composed of 0.21­0.29 helical content, which was comparable to the helical content in the crystal structure of E. coli DegP. The basic/acidic residue ratio of recombinant DegP was 0.56, which was slightly higher than that of DegP from extreme halophiles (average, 0.45) but significantly lower than that of DegP from nonhalophiles (average, 0.94). Conclusions: Recombinant DegP from C. salexigens BKL5 showed proteolytic activity when ß-casein was used as a substrate. In silico analysis indicated that recombinant DegP had characteristics similar to those of halophilic proteins depending on its amino acid composition.

Reversible Activation of Halophilic ß-lactamase from Methanol-Induced Inactive Form: Contrast to Irreversible Inactivation of Non-Halophilic Counterpart.

Effects of a water-miscible organic solvent, methanol, on the structure and activity of halophilic ß-lactamase derived from Chromohalobacter sp.560 (HaBla), were investigated by means of circular dichroism (CD) measurement and enzymatic activity determination. Beta-lactamase activity was enhanced about 1.2-fold in the presence of 10-20% methanol. CD measurement of HaBla revealed different structures depending on the methanol concentration: native-like active form (Form I) in 10-20% methanol and methanol-induced inactive form at higher concentration (Form II in 40-60% and Form III in 75-80% methanol). Incubation of HaBla with 40% methanol led to the complete loss of activity within ~80 min accompanied by the formation of Form II, whose activity was recovered promptly up to ~80% of full activity upon dilution of the methanol concentration to 10%. In addition, when the protein concentration was sufficiently high (e.g., 0.7 mg/ml), HaBla activity of Form III in 75% methanol could be recovered in the same way (with slightly slower recovery rate), upon dilution of the methanol concentration. In contrast, non-halophilic ß-lactamase from Escherichia coli K12 strain MG1655 (EcBla) was irreversibly denatured in the presence of 40% methanol. HaBla showed remarkable ability to renature from the methanol-induced inactive states.

Structure of a highly acidic ß-lactamase from the moderate halophile Chromohalobacter sp. 560 and the discovery of a Cs(+)-selective binding site.

Environmentally friendly absorbents are needed for Sr(2+) and Cs(+), as the removal of the radioactive Sr(2+) and Cs(+) that has leaked from the Fukushima Nuclear Power Plant is one of the most important problems in Japan. Halophilic proteins are known to have many acidic residues on their surface that can provide specific binding sites for metal ions such as Cs(+) or Sr(2+). The crystal structure of a halophilic ß-lactamase from Chromohalobacter sp. 560 (HaBLA) was determined to resolutions of between 1.8 and 2.9 Šin space group P31 using X-ray crystallography. Moreover, the locations of bound Sr(2+) and Cs(+) ions were identified by anomalous X-ray diffraction. The location of one Cs(+)-specific binding site was identified in HaBLA even in the presence of a ninefold molar excess of Na(+) (90 mM Na(+)/10 mM Cs(+)). From an activity assay using isothermal titration calorimetry, the bound Sr(2+) and Cs(+) ions do not significantly affect the enzymatic function of HaBLA. The observation of a selective and high-affinity Cs(+)-binding site provides important information that is useful for the design of artificial Cs(+)-binding sites that may be useful in the bioremediation of radioactive isotopes.

Biochemical characterization of uronate dehydrogenases from three Pseudomonads, Chromohalobacter salixigens, and Polaromonas naphthalenivorans.

Enzyme catalysts will be vital in the development of synthetic biology approaches for converting pectinic monosaccharides from citrus and beet processing waste streams to value-added materials. We describe here the biophysical and mechanistic characterization of uronate dehydrogenases from a wide variety of bacterial sources that convert galacturonic acid, the predominate building block of pectin from these plant sources, and glucuronic acid to their corresponding dicarboxylic acids galactarate and glucarate, the latter being a DOE top value biochemical from biomass. The enzymes from Pseudomonas syringae and Polaromonas naphthalenivorans were found to have the highest reported kcat(glucuronic acid) values, on the order of 220-270 s(-1). The thermal stability of this enzyme type is described for the first time here, where it was found that the Kt((0.5)) value range was >20 °C, and the enzyme from Chromohalobacter was moderately thermostable with Kt((0.5))=62.2 °C. The binding mechanism for these bi-substrate enzymes was also investigated in initial rate experiments, where a predominately steady-state ordered binding pattern was indicated.

Investigating the physiological roles of low-efficiency D-mannonate and D-gluconate dehydratases in the enolase superfamily: pathways for the catabolism of L-gulonate and L-idonate.

The sequence/function space in the D-mannonate dehydratase subgroup (ManD) of the enolase superfamily was investigated to determine how enzymatic function diverges as sequence identity decreases [Wichelecki, D. J., et al. (2014) Biochemistry 53, 2722-2731]. That study revealed that members of the ManD subgroup vary in substrate specificity and catalytic efficiency: high-efficiency (kcat/KM = 10(3)-10(4) M(-1) s(-1)) for dehydration of D-mannonate, low-efficiency (kcat/KM = 10-10(2) M(-1) s(-1)) for dehydration of D-mannonate and/or D-gluconate, and no activity. Characterization of high-efficiency members revealed that these are ManDs in the D-glucuronate catabolic pathway {analogues of UxuA [Wichelecki, D. J., et al. (2014) Biochemistry 53, 4087-4089]}. However, the genomes of organisms that encode low-efficiency members of the ManDs subgroup encode UxuAs; therefore, these must have divergent physiological functions. In this study, we investigated the physiological functions of three low-efficiency members of the ManD subgroup and identified a novel physiologically relevant pathway for L-gulonate catabolism in Chromohalobacter salexigens DSM3043 as well as cryptic pathways for L-gulonate catabolism in Escherichia coli CFT073 and L-idonate catabolism in Salmonella enterica subsp. enterica serovar Enteritidis str. P125109. However, we could not identify physiological roles for the low-efficiency members of the ManD subgroup, allowing the suggestion that these pathways may be either evolutionary relics or the starting points for new metabolic potential.

Expression and bioconversion of recombinant m- and p-hydroxybenzoate hydroxylases from a novel moderate halophile, Chromohalobacter sp.

p-Hydroxybenzoate hydroxylase (pobA) and m-hydroxybenzoate hydroxylase (mobA) genes, from the moderate halophile Chromohalobacter sp. HS-2, were expressed and characterized. Solubilities of overexpressed recombinant MobA and PobA were enhanced by the induction of the heat-shock proteins DnaJ and DnaK. Each MobA and PobA maintained stable activity under high NaCl concentrations. V (max) and K (m) values for MobA with m-hydroxybenzoate were 70 µmol min(-1) mg(-1) protein and 81 µM, respectively. Similarly, those of PobA with p-hydroxybenzoate as substrate were 5 µmol min(-1) mg(-1) protein and 129 µM, respectively. The Escherichia coli expression system, including induction of heat shock proteins, was used to convert hydroxybenzoates into protocatechuate (3,4-dihydroxybenzoate) and revealed that resting cells harboring mobA converted 15 mM m-hydroxybenzoate to 15 mM protocatechuate while those harboring pobA converted 50 mM p-hydroxybenzoate to 35 mM protocatechuate at 30 °C, respectively.

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of glucuronic acid dehydrogenase from Chromohalobacter salexigens.

Glucuronic acid dehydrogenase (GluUADH), the product of the Csal-2474 gene from the halophilic bacterium Chromohalobacter salexigens DSM 3043, is an enzyme with potential use in the conversion of glucuronic acid in seaweed biomass to fuels and chemicals. GluUADH is an enzyme that catalyzes the oxidation of glucuronic acid (GluUA) and galacturonic acid (GalUA) and has a preference for NAD(+) rather than NADP(+) as a cofactor. Recombinant GluUADH was crystallized in the presence of 0.2 M calcium acetate, 0.1 M Tris-HCl pH 7.0 and 20% PEG 3000 at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.1 Å. The GluUADH crystal belonged to space group P6(3), with unit-cell parameters a = b = 122.58, c = 150.49 Å, γ = 120°. With one molecule per asymmetric unit, the crystal volume per unit protein weight (V(M)) is 2.78 Å(3) Da(-1). The structure was solved by the single anomalous dispersion method and structure refinement is in progress.

Racemase activity effected by two dehydrogenases in sulfolactate degradation by Chromohalobacter salexigens: purification of (S)-sulfolactate dehydrogenase.

Chromohalobacter salexigens DSM 3043, whose genome has been sequenced, is known to degrade (R,S)-sulfolactate as a sole carbon and energy source for growth. Utilization of the compound(s) was shown to be quantitative, and an eight-gene cluster (Csal_1764-Csal_1771) was hypothesized to encode the enzymes in the degradative pathway. It comprised a transcriptional regulator (SuyR), a Tripartite Tricarboxylate Transporter-family uptake system for sulfolactate (SlcHFG), two sulfolactate dehydrogenases of opposite sulfonate stereochemistry, namely novel SlcC and ComC [(R)-sulfolactate dehydrogenase] [EC 1.1.1.272] and desulfonative sulfolactate sulfo-lyase (SuyAB) [EC 4.4.1.24]. Inducible reduction of 3-sulfopyruvate, inducible SuyAB activity and induction of an unknown protein were detected. Separation of the soluble proteins from induced cells on an anion-exchange column yielded four relevant fractions. Two different fractions reduced sulfopyruvate with NAD(P)H, a third yielded SuyAB activity, and the fourth contained the unknown protein. The latter was identified by peptide-mass fingerprinting as SlcH, the candidate periplasmic binding protein of the transport system. Separated SuyB was also identified by peptide-mass fingerprinting. ComC was partially purified and identified by peptide-mass fingerprinting. The (R)-sulfolactate that ComC produced from sulfopyruvate was a substrate for SuyAB, which showed that SuyAB is (R)-sulfolactate sulfo-lyase. SlcC was purified to homogeneity. This enzyme also formed sulfolactate from sulfopyruvate, but the latter enantiomer was not a substrate for SuyAB. SlcC was obviously ( S)-sulfolactate dehydrogenase.