Broad Substrate Specificity Research Articles

Enzymatic epimerization of monoterpene indole alkaloids in Kratom.

Monoterpene indole alkaloids (MIAs) are a large, structurally diverse class of bioactive natural products. These compounds are biosynthetically derived from a stereoselective Pictet-Spengler condensation that generates a tetrahydro-β-carboline scaffold characterized by a 3 S stereocenter. However, a subset of MIAs contain a non-canonical 3 R stereocenter. Herein, we report the basis for 3 R -MIA biosynthesis in Mitragyna speciosa (Kratom). We discover the presence of the iminium species, 20 S -3-dehydrocorynantheidine, which led us to hypothesize that isomerization of 3 S to 3 R occurs by oxidation and stereoselective reduction downstream of the initial Pictet-Spengler condensation. Isotopologue feeding experiments implicated young leaves and stems as the sites for pathway biosynthesis, facilitating the identification of an oxidase/reductase pair that catalyzes this epimerization. This enzyme pair has broad substrate specificity, suggesting that the oxidase and reductase may be responsible for the formation of many 3 R -MIAs and downstream spirooxindole alkaloids in Kratom. These enzymes allow biocatalytic access to a range of previously inaccessible pharmacologically active compounds.

PHP-Family Diesterase from Novosphingobium with Broad Specificity and High Catalytic Efficiency against Organophosphate Flame-Retardant Derived Diesters.

Organophosphate flame retardants have been widely used in plastic products since the early 2000s. Unfortunately, these compounds leach out of the plastics over time and are carcinogenic, developmental toxins, and endocrine disruptors. Due to the high usage levels and stable nature of the compounds, widespread contamination of the environment has now been observed. Despite their recent introduction into the environment, bacteria from the Sphingomonadaceae family have evolved a three-step hydrolytic pathway to utilize these compounds. The second step in this pathway in Sphingobium sp. TCM1 is catalyzed by Sb-PDE, which is a member of the polymerase and histidinol phosphatase (PHP) family of phosphatases. This enzyme is only the second case of a PHP-family enzyme capable of hydrolyzing phosphodiesters. Bioinformatics analysis has now been used to identify a second PHP diesterase from Novosphingobium sp. EMRT-2 (No-PDE). Kinetic characterization of Sb-PDE and No-PDE with authentic organophosphate flame-retardant diesters demonstrates that these enzymes are true diesterases with more than 1000-fold selectivity for the diesterase activity seen in some cases. Synthesis of a wide array of authentic flame-retardant diesters has allowed the substrate specificity of these enzymes to be determined, and mutagenic analysis of the active site residues has identified key residues that give rise to the high levels of diesterase activity. Despite high sequence identity, No-PDE is found to have a broader substrate specificity against flame-retardant derived diesters, and kcat/Km values greater than 104 M-1 s-1 are seen with the best substrates.

Protein engineering enables Serratia marcescens nuclease A to hydrolyze nucleic acids under high-salt conditions.

For the purpose of removing nucleotide impurities, Serratia marcescens Nuclease A (SmNucA) is widespread used in biopharmaceutical manufacturing, such as production of adeno-associated viral vector, one of the leading gene delivery platforms that features low immunogenicity. However, such utilization of wild-type SmNucA is limited in saline environment. Depending on downstream process development, the ionic strength can be as high as 500mM, at which the potency of wild-type SmNucA plunges. We herein design an SmNucA variant with four Lys mutations, namely HighSalt NucA, to improve nucleic acids binding under high-salt conditions. Km determination and molecular dynamics (MD) simulation implies a new catalytical mechanism adopted by HighSalt NucA and another mutant that harbors four Arg substitutions at the same sites. We thereby conclude that basic-residue mutations on the SmNucA surface stabilize the local conformation in close proximity to the substrate-binding cleft at saline concentration of 500mM. In addition to Lys and Arg mutations, saturation mutagenesis further indicated that certain hydrophobic residues (Ala, Val, Trp, Tyr) and polar residues (Ser, Thr, Asn, Gln) also render SmNucA salt-tolerant, albeit to differing extent. In contrast to activity loss of the wild-type with 400-500mM NaCl, HighSalt NucA maintains broad substrate specificity even under these extreme conditions, which expands its application prospect in the removal of nucleic acid impurities during process development.

Protective Responses of the Intestinal Epithelial Cell Line HT-29 Cells Exposed to Dephosphorylated Salmonella Flagellin [RESEARCH NOTE

This study aimed to describe the effects of Salmonella Typhimurium flagellin (SFL) dephosphorylated by sweet potato purple acid phosphatase (PAP) on the protective responses of the intestinal epithelial cell line HT-29 cells. The enzyme was reported to display a broad substrate specificity for various organic phosphorylated conjugates and phosphoproteins. Dephosphorylation of SFL by sweet potato PAP decreased to 35% in the presence of 0.05 mM vanadate as compared with the negative control (p < 0.05). Intact SFL and the SFL treated with sweet potato PAP did not remarkably induce the activation of caspase-3 in HT-29 cells at all the tested levels of the substrate. Intact SFL maximally induced the release of IL (interleukin)-8 in HT-29 cells at 1000 ng/mL (p < 0.05). However, the SFL treated with the enzyme inhibited the release of IL-8 at over 100 ng/mL of the substrate as compared with intact SFL, resulting in an approximately 8-fold decrease even at 1000 ng/mL (p < 0.05). The SFL treated with the enzyme decreased the activation of the total ERK1/2 in the cells to 1.9 and 1.7–fold at 10 and 1000 ng/mL of the substrate, respectively, as compared with intact SFL (p < 0.05). In conclusion, sweet potato PAP could be a promising tool for controlling excessive inflammation during Salmonella infection in animal husbandry, and the enzyme could be a safe alternative that can overcome the drawbacks of chemotherapy.

Synthetic Lesions with a Fluorescein Carbamoyl Group As Analogs of Bulky Lesions Removable by Nucleotide Excision Repair: A Comparative Study on Properties.

Mammalian nucleotide excision repair (NER), known for its broad substrate specificity, is responsible for removal of bulky lesions from DNA. Over 30 proteins are involved in NER, which includes two distinct pathways: global genome NER and transcription-coupled repair. The complexity of these processes, the use of extended DNA substrates, and the presence of bulky DNA lesions induced by chemotherapy have driven researchers to seek more effective methods by which to assess NER activity, as well as to develop model DNAs that serve as efficient substrates for studying lesion removal. In this work, we conducted a comparative analysis of model DNAs containing bulky lesions. One of these lesions, N-[6-{5(6)-fluoresceinylcarbamoyl}hexanoyl]-3-amino-1,2-propanediol (nFluL), is known to be efficiently recognized and excised by NER. The second lesion, N-[6-{5(6)-fluoresceinylcarbamoyl}]-3-amino-1,2-propanediol (nFluS), has not previously been tested as a substrate for NER. To evaluate the efficiency of lesion excision, a 3'-terminal labeling method was employed to analyze the excision products. The results showed that nFluS is removed approximately twice as efficiently as nFluL. Comparative analyses of the effects of nFluL and nFluS on the geometry and thermal stability of DNA duplexes - combined with spectrophotometric and spectrofluorimetric titrations of these DNAs with complementary strands - were performed next. They revealed that the absence of an extended flexible linker in nFluS alters the interaction of the bulky fluorescein moiety with neighboring nitrogenous bases in double-stranded DNA. This absence is associated with the enhanced efficiency of excision of nFluS, making it a more effective synthetic analog for studying bulky-lesion removal in model DNA substrates.

Microbial consortia driving (ligno)cellulose transformation in agricultural woodchip bioreactors.

Freshwater ecosystems face significant threats from agricultural runoff, which can lead to eutrophication and subsequent degradation of water quality. One solution to mitigate this issue is using denitrifying woodchip bioreactors (WBRs), where microorganisms convert nitrate into nitrogen gases utilizing lignocellulose as a carbon source. Despite the well-documented polysaccharide-degrading strategies in various systems, the mechanisms employed by denitrifying microorganisms in WBRs remain largely unexplored. This study fills a critical knowledge gap by revealing the degrading strategies of denitrifying microbial communities in WBRs. By integrating state-of-the-art techniques, we have identified key microbial drivers including Giesbergeria, Cellulomonas, Azonexus, and UBA5070 (Fibrobacterota) playing significant roles in lignocellulose transformation and showcasing a broad substrate specificity and complex metabolic capability. Our findings advance the understanding of microbial ecology in WBRs and by revealing the enzymatic activities, this research may inform efforts to improve water quality, protect aquatic ecosystems, and reduce greenhouse gas emissions from WBRs.

Selectivity of a Bioreceptor Element of the Sensor Based on an Organosilicon Material and Microorganism Cells

The yeast cells from Ogataea polymorpha, Blastobotrys adeninivorans, and Debaryomyces hansenii were immobilized on an organosilicon material by the sol-gel method, resulting in a hybrid biocatalyst. The catalytic activity of the immobilized mixture of microorganisms was evaluated using it as a bioreceptor element in a biosensor. The proposed biohybrid material, consisting of the yeast association immobilized on the organosilicate sol-gel matrix, exhibited broad substrate specificity. Thus, the immobilization of multiple yeast strains provides a heterogeneous catalyst able to biologically oxidize a wide range of organic compounds commonly found in wastewater and surface water.

Leptospira Leptolysin Contributes to Serum Resistance but Is Not Essential for Acute Infection.

Previous invitro works focusing on virulence determinants of the spirochete Leptospira implicated metalloproteinases as putative contributing factors to the pathogenicity of these bacteria. Those proteins have the capacity to degrade extracellular matrix components (ECM) and proteins of host's innate immunity, notably effectors of the complement system. In this study, we gained further knowledge on the role of leptolysin, one of the leptospiral-secreted metalloproteinases, previously described as having a broad substrate specificity. We demonstrated that a proportion of human patients with mild leptospirosis evaluated in the current study produced antibodies that recognize leptolysin, thus indicating that the protease is expressed during host infection. Using recombinant protein and a knockout mutant strain, Manilae leptolysin-, we determined that leptolysin contributes to Leptospira interrogans serum resistance invitro, likely by proteolysis of complement molecules of the alternative, the classical, the lectin, and the terminal pathways. Furthermore, in a hamster model of infection, the mutant strain retained virulence; however, infected animals had lower bacterial loads in their kidneys. Further studies are necessary to better understand the role and potential redundancy of metalloproteinases on the pathogenicity of this important neglected disease.

A non-catalytic role of IPMK is required for PLCγ1 activation in T cell receptor signaling by stabilizing the PLCγ1-Sam68 complex

BackgroundPhospholipase C gamma 1 (PLCγ1) is an important mediator of the T cell receptor (TCR) and growth factor signaling. PLCγ1 is activated by Src family kinases (SFKs) and produces inositol 1,4,5-triphosphate (InsP3) from phosphatidylinositol 4,5-bisphosphate (PIP2). Inositol polyphosphate multikinase (IPMK) is a pleiotropic enzyme with broad substrate specificity and non-catalytic activities that mediate various functional protein-protein interactions. Therefore, IPMK plays critical functions in key biological events such as cell growth. However, the contribution of IPMK to the activation of PLCγ1 in TCR signaling remains mostly unelucidated. The current study aimed to elucidate the functions of IPMK in TCR signaling and to uncover the mode of IPMK-mediated signaling action in PLCγ1 activation.MethodsConcanavalin A (ConA)-induced acute hepatitis model was established in CD4+ T cell-specific IPMK knockout mice (IPMKΔCD4). Histological analysis was performed to assess hepatic injury. Primary cultures of naïve CD4+ T cells were used to uncover the role of mechanisms of IPMK in vitro. Western blot analysis, quantitative real-time PCR, and flow cytometry were performed to analyze the TCR-stimulation-induced PLCγ1 activation and the downstream signaling pathway in naïve CD4+ T cells. Yeast two-hybrid screening and co-immunoprecipitation were conducted to identify the IPMK-binding proteins and protein complexes.ResultsIPMKΔCD4 mice showed alleviated ConA-induced acute hepatitis. CD4+ helper T cells in these mice showed reduced PLCγ1 Y783 phosphorylation, which subsequently dampens calcium signaling and IL-2 production. IPMK was found to contribute to PLCγ1 activation via the direct binding of IPMK to Src-associated substrate during mitosis of 68 kDa (Sam68). Mechanistically, IPMK stabilizes the interaction between Sam68 and to PLCγ1, thereby promoting PLCγ1 phosphorylation. Interfering this IPMK-Sam68 binding interaction with IPMK dominant-negative peptides impaired PLCγ1 phosphorylation.ConclusionsOur results demonstrate that IPMK non-catalytically promotes PLCγ1 phosphorylation by stabilizing the PLCγ1–Sam68 complex. Targeting IPMK in CD4+ T cells may be a promising strategy for managing immune diseases caused by excessive stimulation of TCR.

Unique Fn3-like biosensor in σI/anti-σI factors for regulatory expression of major cellulosomal scaffoldins in Pseudobacteroides cellulosolvens.

Lignocellulolytic clostridia employ multiple pairs of alternative σ/anti-σ (SigI/RsgI) factors to regulate cellulosomal components for substrate-specific degradation of cellulosic biomass. The current model has proposed that RsgIs use a sensor domain to bind specific extracellular lignocellulosic components and activate cognate SigIs to initiate expression of corresponding cellulosomal enzyme genes, while expression of scaffoldins can be initiated by several different SigIs. Pseudobacteroides cellulosolvens contains the most complex known cellulosome system and the highest number of SigI-RsgI regulons yet discovered. However, the function of many RsgI sensor domains and their relationship with the various enzyme types are not fully understood. Here, we report that RsgI4 from P. cellulosolvens employs a C-terminal module that bears distant similarity to the fibronectin type III (Fn3) domain and serves as the sensor domain. Substrate-binding analysis revealed that the Fn3-like domain of RsgI4 represents a novel carbohydrate-binding module (CBM) that binds to a wide range of polysaccharide types. Structure determination further revealed that the Fn3-like domain belongs to the type B group of CBMs with a predicted concave face for substrate binding. Promoter sequence analysis of cellulosomal genes revealed that SigI4 is responsible for cellulosomal regulation of major scaffoldins rather than enzymes, consistent with the broad substrate specificity of the RsgI4 sensor domain. Notably, scaffoldins are invariably required as cellulosome components regardless of the substrate type. These findings suggest that the intricate cellulosome system of P. cellulosolvens comprises a more elaborate regulation mechanism than other bacteria and thus expands the paradigm of cellulosome regulation.

A distinct co-expressed sulfurtransferase extends the physiological role of mercaptopropionate dioxygenase in Pseudomonas aeruginosa PAO1

Oxidation and assimilation of persulfides in bacteria is often catalyzed by a persulfide dioxygenase and sulfurtransferase in consecutive reactions. Enzymes responsible for the oxidation of persulfides have not been clearly defined in Pseudomonas aeruginosa PAO1. The characterized mercaptopropionate dioxygenase (MDO) in P. aeruginosa PAO1 has been proposed to catalyze the oxidation of 3-mercaptopropionate. However, the physiological role of MDO is uncertain given the expression of a sulfurtransferase (ST) enzyme on the same operon as the thiol dioxygenase. The st gene had a co-occurrence frequency with mdo of 0.94 demonstrating the co-expression and physiological link of the two genes. There are four tandem rhodanese domains in the ST enzyme with two of the domains containing potential catalytic Cys residues (Cys191 and Cys435) capable of forming a persulfide. Only Cys435 was accessible in thiol quantification assays, and results from H/D-X MS analyses further established the accessibility of the domain containing Cys435. Both thiosulfate and mercaptopyruvate served as sulfur donors to the ST enzyme, with Cys435 forming the persulfide intermediate. Kinetic investigations of MDO suggested the enzyme had a broader substrate specificity than previously identified, oxidizing both mercaptopropionate and mercaptopyruvate thiol and persulfide substrates. The results obtained from these investigations provide insight into the overall mechanism and physiological role of the mdo operon in sulfide oxidation and assimilation.

Novel class of aldehyde reductases identified from Scheffersomyces stipitis for detoxification processes in cellulosic ethanol production industry

The increase in industrialization has led to a significant energy crisis, sparking interest in lignocellulosic biomass for fuel ethanol production because of its renewable characteristics. The complex composition of this biomass requires pretreatment to reduce inhibitors like furfural and hydroxymethylfurfural (HMF), which hinder enzymatic hydrolysis and fermentation, ultimately decreasing ethanol yields. This study investigates the detoxification mechanisms of furan aldehydes in Scheffersomyces stipitis, particularly through the upregulation of genes SsOYE2.2, SsOYE2.7, and SsOYE3.1 under furfural and HMF stress. Enzyme characterization determined that SsOye3.3p is the most active enzyme for reducing both compounds using NADPH. Notably, SsOye2.6p showed the highest catalytic efficiency towards furfural, while SsOye2.8p was optimal for HMF. The study also established the optimal temperature and pH for these enzymatic reactions. Importantly, SsOye2.5p displayed broad substrate specificity, indicating its potential in detoxifying various aldehydes in microbial cells. The findings suggest that genes linked to enhanced enzymatic properties were not significantly induced, indicating that S. stipitis has more substantial potential for furan aldehyde detoxification and can be developed as a chassis organism exhibiting furan aldehyde tolerance. These insights facilitate the development of novel enzymes to counter furan aldehyde inhibitors and the creation of furan aldehyde-tolerant strains via genetic engineering.

Laccase: A Green Biocatalyst Offers Immense Potential for Food Industrial and Biotechnological Applications.

Laccase, a multipurpose biocatalyst, is widely distributed across all kingdoms of life and plays a key role in essential biological processes such as lignin synthesis, degradation, and pigment formation. These functions are critical for fungal growth, plant-pathogen interactions, and maintenance of soil health. Due to its broad substrate specificity, multifunctional nature, and environmentally friendly characteristics, laccase is widely employed as a catalyst in various green chemistry initiatives. With its ability to oxidize a diverse range of phenolic and nonphenolic compounds, laccase has also been found to be useful as a food additive and for assessing food quality parameters. Ongoing advancements in research and technology are continually expanding the recognition of laccase's potential to address global environmental, health, and energy challenges. This review aims to provide critical insights into the applications of laccases in the biotechnology and food industry.

Linkage-specific ubiquitin binding interfaces modulate the activity of the chlamydial deubiquitinase Cdu1 towards poly-ubiquitin substrates.

The chlamydial deubiquitinase Cdu1 of the obligate intracellular human pathogenic bacterium Chlamydia trachomatis plays important roles in the maintenance of chlamydial infection. Despite the structural similarities shared with its homologue Cdu2, both DUBs display remarkable differences in their enzymatic activity towards poly-UB chain substrates. Whereas Cdu1 is highly active towards K48- and K63- poly-UB chains, Cdu2 activity is restricted mostly to mono-UB substrates. Here, we shed light on the molecular mechanisms of the differential activity and the substrate specificity of Cdu1 to better understand the cellular processes it is involved in, including infection-related events. We found that the strikingly elevated activity of Cdu1 relative to its paralogue Cdu2 can be attributed to an N-terminally extended α-helix, which has not been observed in Cdu2. Moreover, by employing isothermal titration calorimetry and nuclear magnetic resonance spectroscopy, we demonstrate the differential recognition of K48- and K63-linked poly-UB substrates by Cdu1. Whereas K63-linked poly-UB substrates appear to be recognized by Cdu1 with only two independent ubiquitin interaction sites, up to four different binding interfaces are present for K48-linked ubiquitin chains. Combined, our data suggest that Cdu1 possesses a poly-UB chain directed activity that may enable its function as a multipurpose DUB with a broad substrate specificity.

Genomic analysis and biodesulfurization potential of a new carbon-sulfur bond cleaving Tsukamurella sp. 3OW.

Direct combustion of sulfur-enriched liquid fuel oil causes sulfur oxide emission, which is one of the main contributors to air pollution. Biodesulfurization is a promising and eco-friendly method to desulfurize a wide range of thiophenic compounds present in fuel oil. Previously, numerous bacterial strains from genera such as Rhodococcus, Corynebacterium, Gordonia, Nocardia, Mycobacterium, Mycolicibacterium, Paenibacillus, Shewanella, Sphingomonas, Halothiobacillus, and Bacillus have been reported to be capable of desulfurizing model thiophenic compounds or fossil fuels. In the present study, we report a new desulfurizing bacterium, Tsukamurella sp. 3OW, capable of desulfurization of dibenzothiophene through the carbon-sulfur bond cleavage 4S pathway. The bacterium showed a high affinity for the hydrocarbon phase and broad substrate specificity towards various thiophenic compounds. The overall genome-related index analysis revealed that the bacterium is closely related to Tsukamurella paurometabola species. The genomic pool of strain 3OW contains 57 genes related to sulfur metabolism, including the key dszABC genes responsible for dibenzothiophene desulfurization. The DBT-adapted cells of the strain 3OW displayed significant resilience and viability in elevated concentrations of crude oil. The bacterium showed a 19 and 37% reduction in the total sulfur present in crude and diesel oil, respectively. Furthermore, FTIR analysis indicates that the oil's overall chemistry remained unaltered following biodesulfurization. This study implies that Tsukamurella paurometabola species, previously undocumented in the context of biodesulfurization, has good potential for application in the biodesulfurization of petroleum oils.

Phospholipase A2 group IVD mediates the transacylation of glycerophospholipids and acylglycerols

In mammalian cells, glycerolipids are mainly synthesized using acyl-CoA–dependent mechanisms. The acyl-CoA–independent transfer of fatty acids between lipids, designated as transacylation reaction, represents an additional mechanism for lipid remodeling and synthesis pathways. Here, we demonstrate that human and mouse phospholipase A2 group IVD (PLA2G4D) catalyzes transacylase reactions using both phospholipids and acylglycerols as substrates. In the presence of monoglycerol and diacylglycerol (MAG and DAG), purified PLA2G4D generates DAG and triacylglycerol, respectively. The enzyme also transfers fatty acids between phospholipids and from phospholipids to acylglycerols. Overexpression of PLA2G4D in COS7 cells enhances the incorporation of polyunsaturated fatty acids into triacylglycerol stores and induces the accumulation of lysophospholipids. In the presence of exogenously added MAG, the enzyme strongly increases cellular DAG formation, while MAG levels are decreased. PLA2G4D is not or poorly detectable in commonly used cell lines. It is expressed in keratinocytes, where it is strongly upregulated by proinflammatory cytokines. Pla2g4d-deficient mouse keratinocytes exhibit complex lipidomic changes in response to cytokine treatment, indicating that PLA2G4D is involved in the remodeling of the lipidome under inflammatory conditions. Transcriptomic analysis revealed that PLA2G4D modulates fundamental biological processes including cell proliferation, differentiation, and signaling. Together, our observations demonstrate that PLA2G4D has broad substrate specificity for fatty acid donor and acceptor lipids, allowing the acyl-CoA-independent synthesis of both phospholipids and acylglycerols. Loss-of-function studies indicate that PLA2G4D affects metabolic and signaling pathways in keratinocytes, which is associated with complex lipidomic and transcriptomic alterations.

Engineering arabinose-to-arabitol conversion in industrial Saccharomyces cerevisiae for sugar beet pulp valorization

Arabitol, a promising low-calorie sweetener for the food industry, faces production challenges due to the high costs and the environmental impact of conventional chemical methods, as well as the inefficiency of biological approaches using native arabitol-producing yeasts.The Saccharomyces cerevisiae GRE3 gene encodes an NADPH-dependent aldose reductase, capable of converting various aldoses to their respective sugar alcohols. By leveraging the enzyme’s broad substrate specificity, the simultaneous conversion of xylose and arabinose into xylitol and arabitol, respectively, was achieved. This study showcases the feasibility of using engineered industrial yeast strains, overexpressing the native GRE3 gene, for the one-step conversion of arabinose to arabitol. Engineered strains (PE2–2-pGRE3, PE2–2-pGRE3-XII5, CA11-pGRE3, CA11-pGRE3-XII5, CAT1- pGRE3 and CAT1- pGRE3-XII5) exhibited successful arabinose-to-arabitol conversion, with a 3.5-fold variation in arabitol concentrations across strains. Further enhancement of the best-performing strain through overexpression of the GAL2 gene significantly improved arabinose transport, resulting in a 1.95-fold increase in arabitol productivity. This engineered strain, free of heterologous genes, also demonstrated its potential by converting arabinose-rich sugar-beet pulp hydrolysate into arabitol, reaching a titer of 15 g/L with a yield of 1 g/g. This study represents the first example of arabitol production from agro-food waste using genome-edited S. cerevisiae strains, which contain no foreign genes, as a versatile and efficient host.

Rigidifying Flexible Sites: A Promising Strategy to Improve Thermostability of Lysophospholipase From Pyrococcus abyssi.

High thermostability of the enzymes is one of the distinguishing characteristics that increase their industrial utility. In the current research work, rigidifying the flexible amino acid residues of a lysophospholipase (Pa-LPL) from Pyrococcus abyssi was used as a protein engineering approach to improve its thermostability. A truncated variant of Pa-LPL (t-LPL∆12) was constructed by trimming its 12 amino acid residues (50-61) through overlap extension PCR. The truncated enzyme worked optimally at 65°C and pH 6.5 with remarkable thermostability at 65°C-85°C. In comparison to wild-type Pa-LPL, 5.8 and 1.2-fold increase in half-life (t1/2) of t-LPL∆12 was observed at 65 (optimum temperature) and 95°C, respectively. The activity of t-LPL∆12 was stimulated by 1 mM Cu2+ followed by Ca2+, Ni2+, Co2+, and Mg2+. Both substrate docking and experimental results indicated that the truncated enzyme could hydrolyze a variety of p-nitrophenyl esters. Km, Vmax, and Kcat for enzymatic hydrolysis of p-nitrophenyl butyrate were calculated to be 1 ± 0.087 mM, 1456 ± 36.474 U/mg, and 1.397 × 1011 min-1, respectively. In short, broad substrate specificity and thermostability of t-LPL∆12 are some of the distinctive features that make it an ideal candidate for degumming of vegetable oils.

Catalytic Redundancies and Conformational Plasticity Drives Selectivity and Promiscuity in Quorum Quenching Lactonases.

Several enzymes from the metallo-β-lactamase-like family of lactonases (MLLs) degrade N-acyl L-homoserine lactones (AHLs). They play a role in a microbial communication system known as quorum sensing, which contributes to pathogenicity and biofilm formation. Designing quorum quenching (QQ) enzymes that can interfere with this communication allows them to be used in a range of industrial and biomedical applications. However, tailoring these enzymes for specific communication signals requires a thorough understanding of their mechanisms and the physicochemical properties that determine their substrate specificities. We present here a detailed biochemical, computational, and structural study of GcL, which is a highly proficient and thermostable MLL with broad substrate specificity. We show that GcL not only accepts a broad range of substrates but also hydrolyzes these substrates through at least two different mechanisms. Further, the preferred mechanism appears to depend on both the substrate structure and/or the nature of the residues lining the active site. We demonstrate that other lactonases, such as AiiA and AaL, show similar mechanistic promiscuity, suggesting that this is a shared feature among MLLs. Mechanistic promiscuity has been seen previously in the lactonase/paraoxonase PON1, as well as with protein tyrosine phosphatases that operate via a dual general acid mechanism. The apparent prevalence of this phenomenon is significant from both a biochemical and protein engineering perspective: in addition to optimizing for specific substrates, it may be possible to optimize for specific mechanisms, opening new doors not just for the design of novel quorum quenching enzymes but also of other mechanistically promiscuous enzymes.

Mannosidase-inhibiting iminosugar production by recombinant Corynebacterium glutamicum harboring the 1-deoxynojirimycin biosynthetic gene cluster

The iminosugar class of carbohydrate-active enzyme inhibitors has therapeutic applications in metabolic syndrome conditions, viral infections and cancer. Compared to chemical synthesis, microbial iminosugar production has benefits of cost, sustainability and optimization. In this study, the 1-deoxynojirimycin (DNJ) biosynthetic gene cluster from Bacillus velezensis MBLB0692, and its individual genes, were cloned into Corynebacterium glutamicum (Cg). Characterizations of the encoded aminotransferase GabT1, phosphatase Yktc1, and dehydrogenase GutB1, were performed with purified enzymes and whole cell biocatalysts bearing individual and clustered (TYB) genes. GabT1 showed a variable pattern in its half-reaction with a slow turnover. GutB1 was an alkaline dehydrogenase with a broad substrate specificity and no divalent ion dependency while the zinc-dependent phosphatase Yktc1 had substrate specificity that was both pH- and ion-dependent. The CgYktc1 and CgGutB1 whole cells were viable biocatalysts with wider ranges of substrates than their enzyme counterparts. The CgTYB cells produced mannosidase-inhibiting iminosugars corresponding to mannojirimycin dehydrate (162 m/z) and deoxymannojirimycin (164 m/z). Mannosidase inhibitors have been found to be effective in treating orphan diseases, cancer and viral infections, and their biosynthesis by recombinant C. glutamicum can be optimized for industrial production and novel drug development.