Scrutiny of Metal Ion Binding Sites in Different Alginate Lyases through In Silico Analysis.

Alginate lyases are epitomized as prospective therapeutic mediators for treating Pseudomonas aeruginosa infections, particularly in the cystic fibrosis airway through alginate degradation thereby improving the efficacy of anti-pseudomonal antibiotics. Investigation of metal-binding residues is significant for expounding the ion specificity of an enzyme and will provide a broad understanding of the potential roles of metal ions in enzyme function and stability. However, experimental analysis of metal ion-binding sites in proteins is time consuming and expensive. Concerning the clinical importance of this therapeutic enzyme, the present study was focused on the prediction and characterization of metal ion-binding sites of different alginate lyases reported in the literature through a computational approach using a Metal Ion-Binding Site Prediction and Docking Server. 3D structures of different alginate lyase from different organisms were retrieved, and these retrieved proteins were docked with twelve different metal ions such as Ca2+, Cu2+, Fe3+, Mg2+, Mn2+, Zn2+, Cd2+, Fe2+, Ni2+, Hg2+, Co2+, and Cu+. The binding affinity and interacting amino acids for alginate lyases produced by different microorganisms were compared and analysed. Further analysis on active site residues of reported alginate lyase and subsequent experiments will reveal the function of different metal ions in enhancing or inhibiting the catalysis of alginate lyase and will help in exploiting the enzyme as an efficient therapeutic agent as well as for industrial applications.

Unexpected Reactions of α,ß-Unsaturated Fatty Acids Provide Insight into the Mechanisms of CYP152 Peroxygenases.

CYP152 peroxygenases catalyze decarboxylation and hydroxylation of fatty acids using H2 O2 as cofactor. To understand the molecular basis for the chemo- and regioselectivity of these unique P450 enzymes, we analyze the activities of three CYP152 peroxygenases (OleTJE , P450SPα , P450BSß ) towards cis- and trans-dodecenoic acids as substrate probes. The unexpected 6S-hydroxylation of the trans-isomer and 4R-hydroxylation of the cis-isomer by OleTJE , and molecular docking results suggest that the unprecedented selectivity is due to OleTJE 's preference of C2-C3 cis-configuration. In addition to the common epoxide products, undecanal is the unexpected major product of P450SPα and P450BSß regardless of the cis/trans-configuration of substrates. The combined H218 O2 tracing experiments, MD simulations, and QM/MM calculations unravel an unusual mechanism for Compound I-mediated aldehyde formation in which the active site water derived from H2 O2 activation is involved in the generation of a four-membered ring lactone intermediate. These findings provide new insights into the unusual mechanisms of CYP152 peroxygenases.

Differential Roles of Three Different Upper Pathway meta Ring Cleavage Product Hydrolases in the Degradation of Dibenzo-p-Dioxin and Dibenzofuran by Sphingomonas wittichii Strain RW1.

Sphingomonas wittichii RW1 grows on the two related compounds dibenzofuran (DBF) and dibenzo-p-dioxin (DXN) as the sole source of carbon. Previous work by others (P. V. Bunz, R. Falchetto, and A. M. Cook, Biodegradation 4:171-178, 1993, https://doi/org/10.1007/BF00695119) identified two upper pathway meta cleavage product hydrolases (DxnB1 and DxnB2) active on the DBF upper pathway metabolite 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)-hexa-2,4-dienoate. We took a physiological approach to determine the role of these two enzymes in the degradation of DBF and DXN by RW1. Single knockouts of either plasmid-located dxnB1 or chromosome-located dxnB2 had no effect on RW1 growth on either DBF or DXN. However, a double-knockout strain lost the ability to grow on DBF but still grew normally on DXN, demonstrating that DxnB1 and DxnB2 are the only hydrolases involved in the DBF upper pathway. Using a transcriptomics-guided approach, we identified a constitutively expressed third hydrolase encoded by the chromosomally located SWIT0910 gene. Knockout of SWIT0910 resulted in a strain that no longer grows on DXN but still grows normally on DBF. Thus, the DxnB1 and DxnB2 hydrolases function in the DBF but not the DXN catabolic pathway, and the SWIT0190 hydrolase functions in the DXN but not the DBF catabolic pathway. IMPORTANCE S. wittichii RW1 is one of only a few strains known to grow on DXN as the sole source of carbon. Much of the work deciphering the related RW1 DXN and DBF catabolic pathways has involved genome gazing, transcriptomics, proteomics, heterologous expression, and enzyme purification and characterization. Very little research has utilized physiological techniques to precisely dissect the genes and enzymes involved in DBF and DXN degradation. Previous work by others identified and extensively characterized two RW1 upper pathway hydrolases. Our present work demonstrates that these two enzymes are involved in DBF but not DXN degradation. In addition, our work identified a third constitutively expressed hydrolase that is involved in DXN but not DBF degradation. Combined with our previous work (T. Y. Mutter and G. J. Zylstra, Appl Environ Microbiol 87:e02464-20, 2021, https://doi.org/10.1128/AEM.02464-20), this means that the RW1 DXN upper pathway involves genes from three very different locations in the genome, including an initial plasmid-encoded dioxygenase and a ring cleavage enzyme and hydrolase encoded on opposite sides of the chromosome.

Separate Upper Pathway Ring Cleavage Dioxygenases Are Required for Growth of Sphingomonas wittichii Strain RW1 on Dibenzofuran and Dibenzo-p-Dioxin.

Sphingomonas wittichii RW1 is one of a few strains known to grow on the related compounds dibenzofuran (DBF) and dibenzo-p-dioxin (DXN) as the sole source of carbon. Previous work by others (B. Happe, L. D. Eltis, H. Poth, R. Hedderich, and K. N. Timmis, J Bacteriol 175:7313-7320, 1993, https://doi.org/10.1128/jb.175.22.7313-7320.1993) showed that purified DbfB had significant ring cleavage activity against the DBF metabolite trihydroxybiphenyl but little activity against the DXN metabolite trihydroxybiphenylether. We took a physiological approach to positively identify ring cleavage enzymes involved in the DBF and DXN pathways. Knockout of dbfB on the RW1 megaplasmid pSWIT02 results in a strain that grows slowly on DBF but normally on DXN, confirming that DbfB is not involved in DXN degradation. Knockout of SWIT3046 on the RW1 chromosome results in a strain that grows normally on DBF but that does not grow on DXN, demonstrating that SWIT3046 is required for DXN degradation. A double-knockout strain does not grow on either DBF or DXN, demonstrating that these are the only ring cleavage enzymes involved in RW1 DBF and DXN degradation. The replacement of dbfB by SWIT3046 results in a strain that grows normally (equal to the wild type) on both DBF and DXN, showing that promoter strength is important for SWIT3046 to take the place of DbfB in DBF degradation. Thus, both dbfB- and SWIT3046-encoded enzymes are involved in DBF degradation, but only the SWIT3046-encoded enzyme is involved in DXN degradation.IMPORTANCES. wittichii RW1 has been the subject of numerous investigations, because it is one of only a few strains known to grow on DXN as the sole carbon and energy source. However, while the genome has been sequenced and several DBF pathway enzymes have been purified, there has been very little research using physiological techniques to precisely identify the genes and enzymes involved in the RW1 DBF and DXN catabolic pathways. Using knockout and gene replacement mutagenesis, our work identifies separate upper pathway ring cleavage enzymes involved in the related catabolic pathways for DBF and DXN degradation. The identification of a new enzyme involved in DXN biodegradation explains why the pathway of DBF degradation on the RW1 megaplasmid pSWIT02 is inefficient for DXN degradation. In addition, our work demonstrates that both plasmid- and chromosomally encoded enzymes are necessary for DXN degradation, suggesting that the DXN pathway has only recently evolved.

Effect of active-site aromatic residues Tyr or Phe on activity and stability of glucose 6-phosphate dehydrogenase from psychrophilic Arctic bacterium Sphingomonas sp.

Cold-adapted enzymes maintain correct conformation at their active sites despite their intrinsically flexible structures. The psychrophilic Arctic bacterium Sphingomonas sp. PAMC 26621 has two glucose 6-phosphate dehydrogenase (G6PD) isozymes, SpG6PD1 involved in the Entner-Doudoroff pathway and SpG6PD2 in the oxidative pentose phosphate pathway. Structural modeling of SpG6PD1 showed that the hydroxyl group of Tyr177 participates in substrate binding by forming a hydrogen bond with the phosphate group of glucose 6-phosphate, whereas in SpG6PD2, a Phe residue is present in the corresponding position of Tyr177. In this study, we investigated how subtle differences in aromatic residues in the substrate-binding pocket of SpG6PD1 affect enzymatic activity and stability. Mutations of Tyr177 to Ala, His, Phe, and Trp caused increases in the rigidity of the SpG6PD1 structure. Particularly, mutants Y177F and Y177W showed increased thermal stabilities compared to wild-type (WT) but 3- and 15-fold lower catalytic efficiencies, respectively. However, mutants Y177A and Y177H became heat-labile at moderate temperatures. These results indicate that an aromatic residue (Tyr or Phe) is necessary for the substrate-binding pocket of SpG6PD1; Tyr with its hydroxyl group is preferred for enzymatic activity, whereas the more hydrophobic Phe is preferred for thermal stability. Substitutions of bulky Trp for Tyr or Phe at this position resulted in substantial loss of activity. Our study suggests that delicate adjustment of aromatic residues can regulate the activity and stability of psychrophilic G6PD isozymes involved in different metabolic pathways.

The Function of ß-1,4-Glucuronosyltransferase WelK in the Sphingan WL Gum Biosynthesis Process in Marine Sphingomonas sp. WG.

The marine-derived polysaccharide WL gum produced by Sphingomonas sp. WG showed commercial utility potential in ink, food, and oil industries. A ß-1,4-glucuronosyltransferase WelK was predicted to catalyze the transfer of glucuronic acid from UDP-glucuronic acid to glucosyl-α-pyrophosphorylpolyprenol intermediate in the WL gum biosynthesis process. Its function was evaluated by bioinformatical analysis, gene knocking out, and overexpressing strategies. Compared to the wild strain, the WL gum production and broth viscosity of the mutant ∆welK were decreased by 71.5% and 99.2% when cultured for 48 h. The gene disruption led to the failure of product preparation. Homologous expression of welK in the native organism can effectively improve WL gum production. When glucose concentration was 6.7%, the WL gum production by the welK-overexpressing strain cultured for 60 h and 84 h reached 32.65 and 43.13 g/L, 134.1%, and 114% of the wild strain. The polysaccharide composition and qRT-PCR analysis showed that the glucuronic acid content was closely related to the expression level of welK. Thus, WelK was proved to play a critical role in the WL gum synthesis and will be an attractive target for metabolic engineering. Our experiment provided a genetic manipulation method for the functional characterization of genes in Sphingomonas sp. WG.

A (6-4)-photolyase from the Antarctic bacterium Sphingomonas sp. UV9: recombinant production and in silico features.

Photolyases are proteins that enzymatically repair the UV-induced DNA damage by a protein-DNA electron transfer mechanism. They repair either cyclobutane pyrimidine dimers or pyrimidine (6-4) pyrimidone photoproducts or just (6-4)-photoproducts. In this work, we report the production and partial characterization of a recombinant (6-4)-photolyase (SphPhrB97) from a bacterial psychrotolerant Antarctic isolate identified as Sphingomonas sp. strain UV9. The spectrum analysis and the in silico study of SphPhrB97 suggest that this enzyme has similar features as compared to the (6-4)-photolyase from Agrobacterium tumefaciens (4DJA; PhrB), including the presence of three cofactors: FAD, DMRL (6,7-dimethyl-8-(1'-D-ribityl) lumazine), and an Fe-S cluster. The homology model of SphPhrB97 predicts that the DNA-binding pocket (area and volume) is larger as compared to (6-4)-photolyases from mesophilic microbes. Based on sequence comparison and on the homology model, we propose an electron transfer pathway towards the FAD cofactor involving the residues Trp342, Trp390, Tyr40, Tyr391, and Tyr399. The phylogenetic tree performed using curated and well-characterized prokaryotic (6-4)-photolyases suggests that SphPhrB97 may have an ancient evolutionary origin. The results suggest that SphPhrB97 is a cold-adapted enzyme, ready to cope with the UV irradiation stress found in a hostile environment, such as Antarctica.

Purification and characterization of a novel medium-chain ribitol dehydrogenase from a lichen-associated bacterium Sphingomonas sp.

Sugar alcohols (polyols) are abundant carbohydrates in lichen-forming algae and transported to other lichen symbionts, fungi, and bacteria. Particularly, ribitol is an abundant polyol in the lichen Cetraria sp. Polyols have important physiological roles in lichen symbiosis, but polyol utilization in lichen-associated bacteria has been largely unreported. Herein, we purified and characterized a novel ribitol dehydrogenase (RDH) from a Cetraria sp.-associated bacterium Sphingomonas sp. PAMC 26621 grown on a minimal medium containing D-ribitol (the RDH hereafter referred to as SpRDH). SpRDH is present as a trimer in its native form, and the molecular weight of SpRDH was estimated to be 39 kDa by SDS-PAGE and 117 kDa by gel filtration chromatography. SpRDH converted D-ribitol to D-ribulose using NAD+ as a cofactor. As far as we know, SpRDH is the first RDH belonging to the medium-chain dehydrogenase/reductase family. Multiple sequence alignments indicated that the catalytic amino acid residues of SpRDH consist of Cys37, His65, Glu66, and Glu157, whereas those of short-chain RDHs consist of Ser, Tyr, and Lys. Furthermore, unlike other short-chain RDHs, SpRDH did not require divalent metal ions for its catalytic activity. Despite SpRDH originating from a psychrophilic Arctic bacterium, Sphingomonas sp., it had maximum activity at 60°C and exhibited high thermal stability within the 4-50°C range. Further studies on the structure/function relationship and catalytic mechanism of SpRDH will expand our understanding of its role in lichen symbiosis.

A natural occurring bifunctional CPD/(6-4)-photolyase from the Antarctic bacterium Sphingomonas sp. UV9.

Photolyases are flavoproteins that repair ultraviolet-induced DNA lesions (cyclobutane pyrimidine dimer or CPD, and pyrimidine (6-4) pyrimidone photoproducts or (6-4)-PPs), using blue light as an energy source. These enzymes are substrate specific, meaning that a specific photolyase repairs either a CPD or a (6-4)-PP. In this work, we produced a class II CPD-photolyase (called as PhrSph98) from the Antarctic bacterium Sphingomonas sp. UV9 by recombinant DNA technology and we purified the enzyme using immobilized metal affinity chromatography. By using an immunochemistry assay, with monoclonal antibodies against CPD and (6-4)-PP, we found that PhrSph98 repairs both DNA lesions. The result was confirmed by immunocytochemistry using immortalized non-tumorigenic human keratinocytes. Results from structure prediction, pocket computation, and molecular docking analyses showed that PhrSph98 has the two expected protein domains (light-harvesting antenna and a catalytic domain), a larger catalytic site as compared with photolyases produced by mesophilic organisms, and that both substrates fit the catalytic domain. The results obtained from predicted homology modeling suggest that the electron transfer pathway may occur following this pathway: Y389-W369-W390-F376-W381/FAD. The evolutionary reconstruction of PhrSph98 suggests that this is a missing link that reflects the transition of (6-4)-PP repair into the CPD repair ability for the class II CPD-photolyases. To the best of our knowledge, this is the first report of a naturally occurring bifunctional, CPD and (6-4)-PP, repairing enzyme. KEY POINTS: • We report the first described bifunctional CPD/(6-4)-photoproducts repairing enzyme. The bifunctional enzyme reaches the nuclei of keratinocyte and repairs the UV-induced DNA damage. The enzyme should be a missing link from an evolutionary point of view. The enzyme may have potential uses in the pharmaceutical and cosmetic industries.

Semi-rational approach to expand the Acyl-CoA Chain length tolerance of Sphingomonas paucimobilis serine palmitoyltransferase.

Serine palmitoyltransferase (SPTase), the first enzyme of the sphingolipid biosynthesis pathway, produces 3-ketodihydrosphingosine by a Claisen-like condensation/decarboxylation reaction of l-Ser and palmitoyl-CoA (n-C16-CoA). Previous structural analysis of Sphingomonas paucimobilis SPTase (SpSPTase) revealed a dynamic active site loop (RPPATP; amino acids 378-383) in which R378 (underlined) forms a salt bridge with the carboxylic acid group of the PLP : l-Ser external aldimine. We hypothesized that this interaction might play a key role in acyl group substrate selectivity and therefore performed site-saturation mutagenesis at position 378 based on semi-rational design to expand tolerance for shorter acyl-CoA's. The resulting library was initially screened for the reaction between l-Ser and dodecanoyl-CoA (n-C12-CoA). The most interesting mutant (R378 K) was then purified and compared to wild-type SpSPTase against a panel of acyl-CoA's. These data showed that the R378 K substitution shifted the acyl group preference to shorter chain lengths, opening the possibility of using this and other engineered variants for biocatalytic C-C bond-forming reactions.

Control of microenvironment around enzymes by hydrogels.

We prepared enzyme-immobilized hydrogels and investigated the effects of the cross-linking density and polymer properties on their oxidation reaction rate. The oxidation rate of enzyme-immobilized hydrogels increased as the cross-linking density in the hydrogels increased. In addition, we controlled the oxidation rate using hydrogels exhibiting an appropriate interaction with a decoy molecule in the hydrogel.

Characterization of alkaline phosphatase PhoK from Sphingomonas sp. BSAR-1 for phosphate monoester synthesis and hydrolysis.

The biocatalytic activity of a so far underexploited alkaline phosphatase, PhoK from Sphingomonas sp. BSAR-1, was extensively studied in transphosphorylation and hydrolysis reactions. The use of high-energy phosphate donors and oligophosphates as suitable phosphate donors was evaluated, as well as the hydrolytic activity on a variety of phosphate monoesters. While substrates bearing free hydroxy group displayed only moderate reactivity as acceptors for transphosphorylation by PhoK, strong hydrolytic activity on a broad variety of phosphate monoesters under alkaline conditions was observed. Site-directed mutagenesis of selected amino acid residues in the active site provided valuable insights on their involvement in enzyme catalysis. The key residue Thr89 so far postulated to engage in enzyme phosphorylation was confirmed to be crucial for catalysis and could be replaced by serine, albeit with much lower catalytic efficiency.

Catalytic Mechanism of Aryl-Ether Bond Cleavage in Lignin by LigF and LigG.

Given the abundance of lignin in nature, multiple enzyme systems have been discovered to cleave the ß-O-4 bonds, the most prevalent intermonomer linkage. In particular, stereospecific cleavage of lignin oligomers by glutathione S-transferases (GSTs) has been reported in several sphingomonads. Here, we apply quantum mechanics/molecular mechanics simulations to study the mechanism of two glutathione-dependent enzymes in the ß-aryl ether catabolic pathway of Sphingomonas sp. SYK-6, namely, LigF, a ß-etherase, and LigG, a lyase. For LigF, the free-energy landscape supports a SN2 reaction mechanism, with the monoaromatic leaving group being promptly neutralized upon release. Specific interactions with conserved residues are responsible for stereoselectivity and for activation of the cofactor as a nucleophile. A glutathione conjugate is also released by LigF and serves the substrate of LigG, undergoing a SN2-like reaction, in which Cys15 acts as the nucleophile, to yield the second monoaromatic product. The simulations suggest that the electron-donating substituent at the para-position found in lignin-derived aromatics and the interaction with Tyr217 are essential for reactivity in LigG. Overall, this work deepens the understanding of the stereospecific enzymatic mechanisms in the ß-aryl ether cleavage pathway and reveals key structural features underpinning the ligninolytic activity detected in several sphingomonad GSTs.

Cloning and characterization of a thermostable glutathione reductase from a psychrophilic Arctic bacterium Sphingomonas sp.

Glutathione reductase is an important oxidoreductase that helps maintain redox homeostasis by catalyzing the conversion of glutathione disulfide to glutathione using NADPH as a cofactor. In this study, we cloned and characterized a glutathione reductase (hereafter referred to as SpGR) from Sphingomonas sp. PAMC 26621, an Arctic bacterium. SpGR comprises 449 amino acids, and functions as a dimer. Surprisingly, SpGR exhibits characteristics of thermophilic enzymes, showing optimum activity at 60°C and thermal stability up to 70°C with ∼50% residual activity at 70°C for 2 h. The amino acid composition analysis of SpGR showed a 1.9-fold higher Arg content (6%) and a 2.7-fold lower Lys/Arg ratio (0.75) compared to the Arg content (3.15%) and the Lys/Arg ratio (2.01) of known psychrophilic glutathione reductases. SpGR also exhibits its activity at 4°C, and circular dichroism and fluorescence spectroscopy results indicate that SpGR maintains its secondary and tertiary structures within the temperature range of 4-70°C. Taken together, the results of this study indicate that despite its origin from a psychrophilic bacterium, SpGR has high thermal stability. Our study provides an insight into the role of glutathione reductase in maintaining the reducing power of an Arctic bacterium in a broad range of temperatures.

Identification of functionally important residues and structural features in a bacterial lignostilbene dioxygenase.

Lignostilbene-α,ß-dioxygenase A (LsdA) from the bacterium Sphingomonas paucimobilis TMY1009 is a nonheme iron oxygenase that catalyzes the cleavage of lignostilbene, a compound arising in lignin transformation, to two vanillin molecules. To examine LsdA's substrate specificity, we heterologously produced the dimeric enzyme with the help of chaperones. When tested on several substituted stilbenes, LsdA exhibited the greatest specificity for lignostilbene (kcatapp = 1.00 ± 0.04 × 106 m-1 s-1). These experiments further indicated that the substrate's 4-hydroxy moiety is required for catalysis and that this moiety cannot be replaced with a methoxy group. Phenylazophenol inhibited the LsdA-catalyzed cleavage of lignostilbene in a reversible, mixed fashion (Kic = 6 ± 1 µm, Kiu = 24 ± 4 µm). An X-ray crystal structure of LsdA at 2.3 Å resolution revealed a seven-bladed ß-propeller fold with an iron cofactor coordinated by four histidines, in agreement with previous observations on related carotenoid cleavage oxygenases. We noted that residues at the dimer interface are also present in LsdB, another lignostilbene dioxygenase in S. paucimobilis TMY1009, rationalizing LsdA and LsdB homo- and heterodimerization in vivo A structure of an LsdA·phenylazophenol complex identified Phe59, Tyr101, and Lys134 as contacting the 4-hydroxyphenyl moiety of the inhibitor. Phe59 and Tyr101 substitutions with His and Phe, respectively, reduced LsdA activity (kcatapp) ∼15- and 10-fold. The K134M variant did not detectably cleave lignostilbene, indicating that Lys134 plays a key catalytic role. This study expands our mechanistic understanding of LsdA and related stilbene-cleaving dioxygenases.

Highly specific sophorose ß-glucosidase from Sphingomonas elodea ATCC 31461 for the efficient conversion of stevioside to rubusoside.

Enzyme specificity and particularity is needed not only in enzymatic separation methods, but also in enzymatic determination methods for plant compound extraction. Stevioside, rubusoside, and rebaudioside A are natural sweet compounds from plants. These compounds have the same skeleton and only contain different side-chain glucosyl groups, making them difficult to separate. However, enzymes that target diterpenoid compounds and show specific activity for side-chain glucosyl groups are rare. Herein, we report the identification and characterization of an enzyme that can target both diterpenoid compounds and sophorose, namely, ß-glucosidase SPBGL1 from Sphingomonas elodea ATCC 31461. SPBGL1 displayed high specificity toward sophorose, and activity toward stevioside, but not rebaudioside A. The stevioside conversion rate was 98%. SPBGL1 also operated at high substrate concentrations, such as in 50% crude steviol glycoside extract. Glucose liberated from stevioside was easy to quantify using the glucose oxidase method, allowing the stevioside content to be determined.

Molecular Mechanisms of Interactions between Monolayered Transition Metal Dichalcogenides and Biological Molecules.

Single layered two-dimensional (2D) materials such as transition metal dichalcogenides (TMDs) show great potential in many microelectronic or nanoelectronic applications. For example, because of extremely high sensitivity, TMD-based biosensors have become promising candidates for next-generation label-free detection. However, very few studies have been conducted on understanding the fundamental interactions between TMDs and other molecules including biological molecules, making the rational design of TMD-based sensors (including biosensors) difficult. This study focuses on the investigations of the fundamental interactions between proteins and two widely researched single-layered TMDs, MoS2, and WS2 using a combined study with linear vibrational spectroscopy attenuated total reflectance FTIR and nonlinear vibrational spectroscopy sum frequency generation vibrational spectroscopy, supplemented by molecular dynamics simulations. It was concluded that a large surface hydrophobic region in a relatively flat location on the protein surface is required for the protein to adsorb onto a monolayered MoS2 or WS2 surface with preferred orientation. No disulfide bond formation between cysteine groups on the protein and MoS2 or WS2 was found. The conclusions are general and can be used as guiding principles to engineer proteins to attach to TMDs. The approach adopted here is also applicable to study interactions between other 2D materials and biomolecules.

Glycoside Hydrolase Family 39 ß-Xylosidases Exhibit ß-1,2-Xylosidase Activity for Transformation of Notoginsenosides: A New EC Subsubclass.

ß-1,2-Xylosidase activity has not been recorded as an EC subsubclass. In this study, phylogenetic analysis and multiple sequence alignments revealed that characterized ß-xylosidases of glycoside hydrolase family (GH) 39 were classified into the same subgroup with conserved amino acid residue positions participating in substrate recognition. Protein-ligand docking revealed that seven of these positions were probably essential to bind xylose-glucose, which is linked by a ß-1,2-glycosidic bond. Amino acid residues in five of the seven positions are invariant, while those in two of the seven positions are variable with low frequency. Both the wild-type ß-xylosidase rJB13GH39 and its mutants with mutation at the two positions exhibited ß-1,2-xylosidase activity, as they hydrolyzed o-nitrophenyl-ß-d-xylopyranoside and transformed notoginsenosides R1 and R2 to ginsenosides Rg1 and Rh1, respectively. The results suggest that all of these characterized GH 39 ß-xylosidases probably show ß-1,2-xylosidase activity, which should be assigned an EC number with these ß-xylosidases as representatives.

Cloning, Expression, and Characterization of a Psychrophilic Glucose 6-Phosphate Dehydrogenase from Sphingomonas sp. PAMC 26621.

Glucose 6-phosphate dehydrogenase (G6PD) (EC 1.1.1.363) is a crucial regulatory enzyme in the oxidative pentose phosphate pathway that provides reductive potential in the form of NADPH, as well as carbon skeletons for the synthesis of macromolecules. In this study, we report the cloning, expression, and characterization of G6PD (SpG6PD1) from a lichen-associated psychrophilic bacterium Sphingomonas sp. PAMC 26621. SpG6PD1 was expressed in Escherichia coli as a soluble protein, having optimum activity at pH 7.5⁻8.5 and 30 °C for NADP⁺ and 20 °C for NAD⁺. SpG6PD1 utilized both NADP⁺ and NAD⁺, with the preferential utilization of NADP⁺. A high Km value for glucose 6-phosphate and low activation enthalpy (ΔH‡) compared with the values of mesophilic counterparts indicate the psychrophilic nature of SpG6PD1. Despite the secondary structure of SpG6PD1 being maintained between 4⁻40 °C, its activity and tertiary structure were better preserved between 4⁻20 °C. The results of this study indicate that the SpG6PD1 that has a flexible structure is most suited to a psychrophilic bacterium that is adapted to a permanently cold habitat.

Use of isotopically labeled substrates reveals kinetic differences between human and bacterial serine palmitoyltransferase.

Isotope labels are frequently used tools to track metabolites through complex biochemical pathways and to discern the mechanisms of enzyme-catalyzed reactions. Isotopically labeled l-serine is often used to monitor the activity of the first enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT), as well as labeling downstream cellular metabolites. Intrigued by the effect that isotope labels may be having on SPT catalysis, we characterized the impact of different l-serine isotopologues on the catalytic activity of recombinant SPT isozymes from humans and the bacterium Sphingomonas paucimobilis Our data show that S. paucimobilis SPT activity displays a clear isotope effect with [2,3,3-D]l-serine, whereas the human SPT isoform does not. This suggests that although both human and S. paucimobilis SPT catalyze the same chemical reaction, there may well be underlying subtle differences in their catalytic mechanisms. Our results suggest that it is the activating small subunits of human SPT that play a key role in these mechanistic variations. This study also highlights that it is important to consider the type and location of isotope labels on a substrate when they are to be used in in vitro and in vivo studies.