Structural and Biochemical Insights into Bis(2-hydroxyethyl) Terephthalate Degrading Carboxylesterase Isolated from Psychrotrophic Bacterium Exiguobacterium antarcticum.

This study aimed to elucidate the crystal structure and biochemically characterize the carboxylesterase EaEst2, a thermotolerant biocatalyst derived from Exiguobacterium antarcticum, a psychrotrophic bacterium. Sequence and phylogenetic analyses showed that EaEst2 belongs to the Family XIII group of carboxylesterases. EaEst2 has a broad range of substrate specificities for short-chain p-nitrophenyl (pNP) esters, 1-naphthyl acetate (1-NA), and 1-naphthyl butyrate (1-NB). Its optimal pH is 7.0, losing its enzymatic activity at temperatures above 50 °C. EaEst2 showed degradation activity toward bis(2-hydroxyethyl) terephthalate (BHET), a polyethylene terephthalate degradation intermediate. We determined the crystal structure of EaEst2 at a 1.74 Å resolution in the ligand-free form to investigate BHET degradation at a molecular level. Finally, the biochemical stability and immobilization of a crosslinked enzyme aggregate (CLEA) were assessed to examine its potential for industrial application. Overall, the structural and biochemical characterization of EaEst2 demonstrates its industrial potency as a biocatalyst.

Feruloyl Esterase (LaFae) from Lactobacillus acidophilus: Structural Insights and Functional Characterization for Application in Ferulic Acid Production.

Ferulic acid and related hydroxycinnamic acids, used as antioxidants and preservatives in the food, cosmetic, pharmaceutical and biotechnology industries, are among the most abundant phenolic compounds present in plant biomass. Identification of novel compounds that can produce ferulic acid and hydroxycinnamic acids, that are safe and can be mass-produced, is critical for the sustainability of these industries. In this study, we aimed to obtain and characterize a feruloyl esterase (LaFae) from Lactobacillus acidophilus. Our results demonstrated that LaFae reacts with ethyl ferulate and can be used to effectively produce ferulic acid from wheat bran, rice bran and corn stalks. In addition, xylanase supplementation was found to enhance LaFae enzymatic hydrolysis, thereby augmenting ferulic acid production. To further investigate the active site configuration of LaFae, crystal structures of unliganded and ethyl ferulate-bound LaFae were determined at 2.3 and 2.19 Å resolutions, respectively. Structural analysis shows that a Phe34 residue, located at the active site entrance, acts as a gatekeeper residue and controls substrate binding. Mutating this Phe34 to Ala produced an approximately 1.6-fold increase in LaFae activity against p-nitrophenyl butyrate. Our results highlight the considerable application potential of LaFae to produce ferulic acid from plant biomass and agricultural by-products.

Investigating the Functional Role of the Cysteine Residue in Dehydrin from the Arctic Mouse-Ear Chickweed Cerastium arcticum.

The stress-responsive, SK5 subclass, dehydrin gene, CaDHN, has been identified from the Arctic mouse-ear chickweed Cerastium arcticum. CaDHN contains an unusual single cysteine residue (Cys143), which can form intermolecular disulfide bonds. Mutational analysis and a redox experiment confirmed that the dimerization of CaDHN was the result of an intermolecular disulfide bond between the cysteine residues. The biochemical and physiological functions of the mutant C143A were also investigated by in vitro and in vivo assays using yeast cells, where it enhanced the scavenging of reactive oxygen species (ROS) by neutralizing hydrogen peroxide. Our results show that the cysteine residue in CaDHN helps to enhance C. arcticum tolerance to abiotic stress by regulating the dimerization of the intrinsically disordered CaDHN protein, which acts as a defense mechanism against extreme polar environments.

Structural and functional analysis of a dimeric fumarylacetoacetate hydrolase (EaFAH) from psychrophilic Exiguobacterium antarcticum.

Fumarylacetoacetate hydrolase (FAH) is essential for the degradation of aromatic amino acids as well as for the cleavage of carbon-carbon bonds in metabolites or small organic compounds. Here, the X-ray crystal structure of EaFAH, a dimeric fumarylacetoacetate hydrolase from Exiguobacterium antarcticum, was determined, and its functional properties were investigated using biochemical methods. EaFAH adopts a mixed ß-sandwich roll fold with a highly flexible lid region (Val73-Leu94), and an Mg2+ ion is bound at the active site by coordinating to the three carboxylate oxygen atoms of Glu124, Glu126, and Asp155. The hydrolytic activity of EaFAH toward various substrates, including linalyl acetate was investigated using native polyacrylamide gel electrophoresis, activity staining, gel filtration, circular dichroism spectroscopy, fluorescence, and enzyme assays.

Characterization and mutation anaylsis of a cold-active bacterial hormone-sensitive lipase from Salinisphaera sp. P7-4.

In mammals, hormone sensitive lipase (EC 3.1.1.79, HSL) catalyzes the hydrolysis of triacylglycerols as well as the modifications of a broad range of hydrophobic substrates containing ester linkages. HSLs are composed of an N-terminal ligand-binding domain and a C-terminal catalytic domain. Bacterial hormone-sensitive lipases (bHSLs), which are homologous to the C-terminal domain of mammalian HSLs, have a catalytic triad composed of Ser, His, and Asp. Here, a novel cold-active hormone-sensitive lipase (SaHSL) from Salinisphaera sp. P7-4 was identified, functionally characterized, and subjected to site-directed mutations. The enzymatic properties of SaHSL were investigated using several biochemical and biophysical methods. Interestingly, SaHSL exhibited the ability to act on a broad range of substrates including glyceryl tributyrate and glucose pentaacetate. Homology modeling and site-directed mutagenesis indicated that hydrophobic residues (Leu156, Phe164, and Val204) around the substrate-binding pocket were involved in substrate recognition. In addition, highly conserved amino acids (Glu201, Arg207, Leu208, and Asp227) in the regulatory regions were found to be responsible for substrate specificity, thermostability, and enantioselectivity. In summary, this work provides new insights into the understanding of the C-terminal domain of HSL family and evidence that SaHSL can be used in a wide range of industrial applications.

Structural and functional characterization of a novel cold-active S-formylglutathione hydrolase (SfSFGH) homolog from Shewanella frigidimarina, a psychrophilic bacterium.

BACKGROUND: S-Formylglutathione is hydrolyzed to glutathione and formate by an S-formylglutathione hydrolase (SFGH) (3.1.2.12). This thiol esterase belongs to the esterase family and is also known as esterase D. SFGHs contain highly conserved active residues of Ser-Asp-His as a catalytic triad at the active site. Characterization and investigation of SFGH from Antarctic organisms at the molecular level is needed for industrial use through protein engineering. RESULTS: A novel cold-active S-formylglutathione hydrolase (SfSFGH) from Shewanella frigidimarina, composed of 279 amino acids with a molecular mass of ~ 31.0 kDa, was characterized. Sequence analysis of SfSFGH revealed a conserved pentapeptide of G-X-S-X-G found in various lipolytic enzymes along with a putative catalytic triad of Ser148-Asp224-His257. Activity analysis showed that SfSFGH was active towards short-chain esters, such as p-nitrophenyl acetate, butyrate, hexanoate, and octanoate. The optimum pH for enzymatic activity was slightly alkaline (pH 8.0). To investigate the active site configuration of SfSFGH, we determined the crystal structure of SfSFGH at 2.32 Å resolution. Structural analysis shows that a Trp182 residue is located at the active site entrance, allowing it to act as a gatekeeper residue to control substrate binding to SfSFGH. Moreover, SfSFGH displayed more than 50% of its initial activity in the presence of various chemicals, including 30% EtOH, 1% Triton X-100, 1% SDS, and 5 M urea. CONCLUSIONS: Mutation of Trp182 to Ala allowed SfSFGH to accommodate a longer chain of substrates. It is thought that the W182A mutation increases the substrate-binding pocket and decreases the steric effect for larger substrates in SfSFGH. Consequently, the W182A mutant has a broader substrate specificity compared to wild-type SfSFGH. Taken together, this study provides useful structure-function data of a SFGH family member and may inform protein engineering strategies for industrial applications of SfSFGH.

Properties of phase transition of ice binding protein from Arctic yeast (LeIBP) utilizing differential scanning calorimetry (DSC) and Raman spectroscopy.

Ice binding proteins (IBPs) have been attracting significant interest on account of their characteristic of inhibiting ice growth and recrystallization. Owing to their unique characteristics, IBPs have been studied for applications in food, pharmaceuticals, and medicine, as well as from a general scientific point of view. In this study, we have used differential scanning calorimetry (DSC) and Raman spectroscopy as tools to understand the ice binding activity of the Arctic-yeast-originating extracellular ice binding glycoprotein (LeIBP) isolated from Leucosporidium sp. AY30. From the DSC results, an increase in the specific heat capacity was confirmed for 1 mg/mL LeIBP, which suggested that additional heat flow was required for the change in temperature. In addition, the temperature corresponding to the phase change of the solution was measured, and Raman spectroscopy was carried out on the frozen and molten phases, respectively. From the results of Raman analysis, we confirmed that the helical vibrations related to the ice binding sites on LeIBP were dramatically suppressed when the LeIBP solution was frozen. Furthermore, principal component analysis (PCA) of the Raman spectra yielded the contrast factor between the freezing and melting states. Both DSC and Raman spectroscopy are widely used to study the ice binding activity and the structural changes associated with molecular vibrations in cryobiology.

Crystal structure of cis-dihydrodiol naphthalene dehydrogenase (NahB) from Pseudomonas sp. MC1: Insights into the early binding process of the substrate.

The bacterial strain Pseudomonas sp. MC1 harbors an 81-kb metabolic plasmid, which encodes enzymes involved in the conversion of naphthalene to salicylate. Of these, the enzyme NahB (cis-dihydrodiol naphthalene dehydrogenase), which catalyzes the second reaction of this pathway, binds to various substrates such as cis-1,2-dihydro-1,2-dihydroxy-naphthalene (1,2-DDN), cis-2,3-dihydro-2,3-dihydroxybiphenyl (2,3-DDB), and 3,4-dihydro-3,4-dihydroxy-2,2',5,5'-tetrachlorobiphenyl (3,4-DD-2,2',5-5-TCB). However, the mechanism underlying its broad substrate specificity is unclear owing to the lack of structural information. Here, we determined the first crystal structures of NahB in the absence and presence of NAD+ and 2,3-dihydroxybiphenyl (2,3-DB). Structure analysis suggests that the flexible substrate-binding loop allows NahB to accommodate diverse substrates. Furthermore, we defined the initial steps of substrate recognition and identified the early substrate-binding site in the substrate recognition process through the complex structure with ligands.

Crystal structures of aldehyde deformylating oxygenase from Limnothrix sp. KNUA012 and Oscillatoria sp. KNUA011.

The cyanobacterial aldehyde deformylating oxygenase (cADO) is a key enzyme that catalyzes the unusual deformylation of aliphatic aldehydes for alkane biosynthesis and can be applied to the production of biofuel in vitro and in vivo. In this study, we determined crystal structures of two ADOs from Limnothrix sp. KNUA012 (LiADO) and Oscillatoria sp. KNUA011 (OsADO). The structures of LiADO and OsADO resembled those of typical cADOs, consisting of eight α-helices found in ferritin-like di-iron proteins. However, structural comparisons revealed that while the LiADO active site was vacant of iron and substrates, the OsADO active site was fully occupied, containing both a coordinated metal ion and substrate. Previous reports indicated that helix 5 is capable of adopting two distinct conformations depending upon the existence of bound iron. We observed that helix 5 of OsADO with an iron bound in the active site presented as a long helix, whereas helix 5 of LiADO, which lacked iron in the active site, presented two conformations (one long and two short helices), indicating that an equilibrium exists between the two states in solution. Furthermore, acquisition of a structure having a fully occupied active site is unique in the absence of higher iron concentrations as compared with other cADO structures, wherein low affinity for iron complicates the acquisition of crystal structures with bound iron. An in-depth analysis of the ADO apo-enzyme, the enzyme with substrate bound, and the enzyme with both iron and substrate bound provided novel insight into substrate-binding modes in the absence of a coordinated metal ion and suggested a separate two-step binding mechanism for substrate and iron co-factors. Moreover, our results provided a comprehensive structural basis for conformational changes induced by binding of the substrate and co-factor.

Biochemical and structural characterization of quinoprotein aldose sugar dehydrogenase from Thermus thermophilus HJ6: Mutational analysis of Tyr156 in the substrate-binding site.

The gene encoding a quinoprotein aldose sugar dehydrogenase (ASD) from Thermus thermophilus HJ6 (Tt_ASD) was cloned and sequenced; it comprised 1059 nucleotides encoding a protein containing 352 amino acids that had a predicted molecular mass of 38.9 kDa. The deduced amino acid sequence showed 42.9% and 33.9% identities to the ASD proteins from Pyrobaculum aerophilum and Escherichia coli, respectively. The biochemical properties of Tt_ASD were characterized. The optimum pH for the oxidation of glucose was 7.0-7.5 and the optimum temperature was 70 °C. The half-life of heat inactivation for the apoenzyme was about 25 min at 85 °C. The enzyme was highly thermostable, and the activity of the pyrroloquinoline quinone-bound holoenzyme was not lost after incubation at 85 °C for 100 min. Tt_ASD could oxidize various sugars, including hexoses, pentoses, disaccharides, and polysaccharides, in addition to alcohols. Structural analysis suggested that Tyr156 would be the substrate-binding residue. Two mutants, Y156A and Y156K, had impaired activities and affinities for all substrates and completely lost their activities for alcohols. This structural and mutational analysis of Tt_ASD demonstrates the crucial role of Tyr156 in determining substrate specificity.

Expression, refolding, and characterization of a small laccase from Thermus thermophilus HJ6.

An open reading frame of the Thermus thermophilus HJ6 hypothetical laccase, which composed of 729 bases, was cloned and expressed as a fusion protein with six histidine residues in Escherichia coli SoluBL21™ cells. The resulting insoluble bodies were separated from cellular debris by centrifugation and solubilized with 6M guanidine HCl. The solubilized protein was refolded by a simple on-column refolding procedure using Ni-chelation affinity chromatography and then the refolded protein was purified by gel filtration chromatography. It showed a single band with a molecular mass of 27kDa in SDS-PAGE. The results from UV-visible absorption and electron paramagnetic resonance (EPR) analysis suggested that the enzyme had the typical copper sites, type-1, 2, and 3 Cu(II) of laccase. The purified enzyme exhibited the laccase activity with the optimal catalytic temperature at 75°C. The optimum pH for the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and syringaldazine was 4.5 and 6.0, respectively. The recombinant protein showed high thermostability, and the half-life of heat inactivation was about 50min at 85°C. The enzyme oxidized various known laccase substrates, its lowest Km value being for syringaldazine, highest kcat value for guaiacol, and highest kcat/Km for 2,6-dimethoxy-phenol. The enzyme reaction was strongly inhibited by the metal chelators and the thiol compounds.

Cold adaptation of a psychrophilic chaperonin from Psychrobacter sp. and its application for heterologous protein expression.

OBJECTIVES: A chaperonin, PsyGroELS, from the Antarctic psychrophilic bacterium Psychrobacter sp. PAMC21119, was examined for its role in cold adaptation when expressed in a mesophilic Escherichia coli strain. RESULTS: Growth of E. coli harboring PsyGroELS at 10 °C was increased compared to the control strain. A co-expression system using PsyGroELS was developed to increase productivity of the psychrophilic enzyme PsyEst9. PsyEst9 was cloned and expressed using three E. coli variants that co-expressed GroELS from PAMC21119, E. coli, or Oleispira antarctica RB8(T). Co-expression with PsyGroELS was more effective for the production of PsyEst9 compared tothe other chaperonins. CONCLUSION: PsyGroELS confers cold tolerance to E. coli, and shows potential as an effective co-expression system for the stable production of psychrophilic proteins.

Enhancing extracellular lipolytic enzyme production in an arctic bacterium, Psychrobacter sp. ArcL13, by using statistical optimization and fed-batch fermentation.

A strain isolated from seawater samples in the Chuckchi Sea and exhibiting extracellular lipolytic activity was identified using 16S rRNA gene sequence analysis as Psychrobacter sp. ArcL13. The lipolytic enzyme exhibited cold-active properties and high hydrolytic activity toward p-nitrophenyl caprylate (C8), p-nitrophenyl decanoate (C10), and sunflower oil. Statistical optimization of the medium components was performed to enhance the production of cold-active extracellular lipolytic activity. Glucose, yeast extract (YE), and NaCl were selected as the main efficient nutrient sources. Fed-batch fermentation using optimized medium with concentrated YE as the main feeding material showed a maximum lipolytic activity of 10.7 U/mL, which was a 21-fold increase in production over unoptimized flask culture conditions. The information obtained in the present study could prove applicable to the production of cold-active lipase on a large scale.

Characterization and a point mutational approach of a psychrophilic lipase from an arctic bacterium, Bacillus pumilus.

A bacterium with lipolytic activity was isolated from the Chukchi Sea within the Arctic Ocean. The lipase BpL5 from the isolate, Bacillus pumilus ArcL5, belongs to subfamily 4 of lipase family I. The optimum pH and temperature of the recombinant enzyme BpL5, as expressed in Escherichia coli, were 9.0 and 20 °C, respectively. The enzyme retained 85 % of its activity at 5 °C. There was a significant difference between temperatures for maximal activity (20 °C) and for protein denaturation (approx. 45 °C). The enzyme preferred middle-chain (C8) p-nitrophenyl substrates. Two mutants, S139A and S139Y, were rationally designed based on the 3D-structure model, and their activities were compared with that of the wild type. The both mutants showed significantly improved activity against tricaprylin.

The role of disulfide bond in hyperthermophilic endocellulase.

The hyperthermophilic endocellulase, EGPh (glycosyl hydrolase family 5) from Pyrococcus horikoshii possesses 4 cysteine residues forming 2 disulfide bonds, as identified by structural analysis. One of the disulfide bonds is located at the proximal region of the active site in EGPh, which exhibits a distinct pattern from that of the thermophilic endocellulase EGAc (glycosyl hydrolase family 5) of Acidothermus cellulolyticus despite the structural similarity between the two endocellulases. The structural similarity between EGPh and EGAc suggests that EGPh possesses a structure suitable for changing the position of the disulfide bond corresponding to that in EGAc. Introduction of this alternative disulfide bond in EGPh, while removing the original disulfide bond, did not result in a loss of enzymatic activity but the EGPh was no longer hyperthermostable. These results suggest that the contribution of disulfide bond to hyperthermostability at temperature higher than 100 °C is restrictive, and that its impact is dependent on the specific structural environment of the hyperthermophilic proteins. The data suggest that the structural position and environment of the disulfide bond has a greater effect on high-temperature thermostability of the enzyme than on the potential energy of the dihedral angle that contributes to disulfide bond cleavage.

Crystal structure and biochemical analysis of acetylesterase (LgEstI) from Lactococcus garvieae.

Esterase, a member of the serine hydrolase family, catalyzes the cleavage and formation of ester bonds with high regio- and stereospecificity, making them attractive biocatalysts for the synthesis of optically pure molecules. In this study, we performed an in-depth biochemical and structural characterization of a novel microbial acetylesterase, LgEstI, from the bacterial fish pathogen Lactococcus garvieae. The dimeric LgEstI displayed substrate preference for the short acyl chain of p-nitrophenyl esters and exhibited increased activity with F207A mutation. Comparative analysis with other esterases indicated that LgEstI has a narrow and shallow active site that may exhibit substrate specificity to short acyl chains. Unlike other esterases, LgEstI contains bulky residues such as Trp89, Phe194, and Trp217, which block the acyl chain channel. Furthermore, immobilized LgEstI retained approximately 90% of its initial activity, indicating its potential in industrial applications. This study expands our understanding of LgEstI and proposes novel ideas for improving its catalytic efficiency and substrate specificity for various applications.

Structural and biochemical insights into PsEst3, a new GHSR-type esterase obtained from Paenibacillus sp. R4.

PsEst3, a psychrophilic esterase obtained from Paenibacillus sp. R4, which was isolated from the permafrost of Alaska, exhibits relatively high activity at low temperatures. Here, crystal structures of PsEst3 complexed with various ligands were generated and studied at atomic resolution, and biochemical studies were performed to analyze the structure-function relationship of PsEst3. Certain unique characteristics of PsEst3 distinct from those of other classes of lipases/esterases were identified. Firstly, PsEst3 contains a conserved GHSRA/G pentapeptide sequence in the GxSxG motif around the nucleophilic serine. Additionally, it contains a conserved HGFR/K consensus sequence in the oxyanion hole, which is distinct from that in other lipase/esterase families, as well as a specific domain composition (for example a helix-turn-helix motif) and a degenerative lid domain that exposes the active site to the solvent. Secondly, the electrostatic potential of the active site in PsEst3 is positive, which may cause unintended binding of negatively charged chemicals in the active site. Thirdly, the last residue of the oxyanion hole-forming sequence, Arg44, separates the active site from the solvent by sealing the acyl-binding pocket, suggesting that PsEst3 is an enzyme that is customized to sense an unidentified substrate that is distinct from those of classical lipases/esterases. Collectively, this evidence strongly suggests that PsEst3 belongs to a distinct family of esterases.

Crystallization and preliminary X-ray analysis of a hyperthermophilic endoglucanase from Pyrococcus furiosus.

The hyperthermophilic glycoside hydrolase family 12 endocellulase from the archaeon Pyrococcus furiosus (EGPf) catalyzes the hydrolytic cleavage of the ß-1,4-glucosidic linkage in ß-glucans in biomass. EGPf (Gene ID PF0854; EC 3.2.1.4) contains a signal sequence and proline- and hydroxyl-rich regions at the N-terminus. Truncated EGPf (EGPfΔN30) without the proline- and hydroxyl-rich regions at the N-terminus was prepared and subjected to crystallization experiments. Crystals were obtained using the hanging-drop vapour-diffusion method at 303 K. An X-ray diffraction data set was collected to 1.07 Å resolution at 100 K. The crystal belonged to space group P2(1)2(1)2, with unit-cell parameters a = 58.01, b = 118.67, c = 46.76 Å. The presence of one molecule of enzyme per asymmetric unit gives a crystal volume per protein mass (V(M)) of 2.63 Å(3) Da(-1) and a solvent content of 53.3%(v/v).

Functional analysis of hyperthermophilic endocellulase from Pyrococcus horikoshii by crystallographic snapshots.

A hyperthermophilic membrane-related ß-1,4-endoglucanase (family 5, cellulase) of the archaeon Pyrococcus horikoshii was found to be capable of hydrolysing cellulose at high temperatures. The hyperthermophilic cellulase has promise for applications in biomass utilization. To clarify its detailed function, we determined the crystal structures of mutants of the enzyme in complex with either the substrate or product ligands. We were able to resolve different kinds of complex structures at 1.65-2.01 Å (1 Å=0.1 nm). The structural analysis of various mutant enzymes yielded a sequence of crystallographic snapshots, which could be used to explain the catalytic process of the enzyme. The substrate position is fixed by the alignment of one cellobiose unit between the two aromatic amino acid residues at subsites +1 and +2. During the enzyme reaction, the glucose structure of cellulose substrates is distorted at subsite -1, and the ß-1,4-glucoside bond between glucose moieties is twisted between subsites -1 and +1. Subsite -2 specifically recognizes the glucose residue, but recognition by subsites +1 and +2 is loose during the enzyme reaction. This type of recognition is important for creation of the distorted boat form of the substrate at subsite -1. A rare enzyme-substrate complex was observed within the low-activity mutant Y299F, which suggested the existence of a trapped ligand structure before the formation by covalent bonding of the proposed intermediate structure. Analysis of the enzyme-substrate structure suggested that an incoming water molecule, essential for hydrolysis during the retention process, might be introduced to the cleavage position after the cellobiose product at subsites +1 and +2 was released from the active site.