Crystal structure of bacterial ubiquitin ADP-ribosyltransferase CteC reveals a substrate-recruiting insertion.

ADP-ribosylation is a post-translational modification involved in regulation of diverse cellular pathways. Interestingly, many pathogens have been identified to utilize ADP-ribosylation as a way for host manipulation. A recent study found that CteC, an effector from the bacterial pathogen Chromobacterium violaceum, hinders host ubiquitin (Ub) signaling pathways via installing mono-ADP-ribosylation on threonine 66 of Ub. However, the molecular basis of substrate recognition by CteC is not well understood. In this article, we probed the substrate specificity of this effector at protein and residue levels. We also determined the crystal structure of CteC in complex with NAD+, which revealed a canonical mono-ADP-ribosyltransferase fold with an additional insertion domain. The AlphaFold-predicted model differed significantly from the experimentally determined structure, even in regions not used in crystal packing. Biochemical and biophysical studies indicated unique features of the NAD+ binding pocket, while showing selectivity distinction between Ub and structurally close Ub-like modifiers and the role of the insertion domain in substrate recognition. Together, this study provides insights into the enzymatic specificities and the key structural features of a novel bacterial ADP-ribosyltransferase involved in host-pathogen interaction.

Transaminases as suitable catalysts for the synthesis of enantiopure ß,ß-difluoroamines.

Transaminases have shown the ability to catalyze the amination of a series of aliphatic and (hetero)aromatic α,α-difluorinated ketones with high stereoselectivity, thus providing the corresponding ß,ß-difluoroamines in high isolated yields (55-82%) and excellent enantiomeric excess (>99%). It was also observed that these activated substrates could be quantitatively transformed by employing a small molar excess of the amine donor since this amination process was thermodynamically favored. Selected transformations could be scaled up to 500 mg, showing the robustness of this methodology.

Direct RBS Engineering of the biosynthetic gene cluster for efficient productivity of violaceins in E. coli.

BACKGROUND: Violaceins have attracted much attention as potential targets used in medicines, food additives, insecticides, cosmetics and textiles, but low productivity was the key factor to limit their large-scale applications. This work put forward a direct RBS engineering strategy to engineer the violacein biosynthetic gene cluster cloned from Chromobacterium violaceum ATCC 12,472 to efficiently improve the fermentation titers. RESULTS: Through four-rounds of engineering of the native RBSs within the violaceins biosynthetic operon vioABCDE, this work apparently broke through the rate-limiting steps of intermediates conversion, resulting in 2.41-fold improvement of violaceins production compared to the titers of the starting strain Escherichia coli BL21(DE3) (Vio12472). Furthermore, by optimizing the batch-fermentation parameters including temperature, concentration of IPTG inducer and fermentation time, the maximum yield of violaceins from (BCDE)m (tnaA-) reached 3269.7 µM at 2 mM tryptophan in the medium. Interestingly, rather than previous reported low temperature (20 ℃), we for the first time found the RBS engineered Escherichia coli strain (BCDE)m worked better at higher temperature (30 ℃ and 37 ℃), leading to a higher-level production of violaceins. CONCLUSIONS: To our knowledge, this is the first time that a direct RBS engineering strategy is used for the biosynthesis of natural products, having the potential for a greater improvement of the product yields within tryptophan hyperproducers and simultaneously avoiding the costly low temperature cultivation for large-scale industrial production of violaciens. This direct RBS engineering strategy could also be easily and helpfully used in engineering the native RBSs of other larger and value-added natural product biosynthetic gene clusters by widely used site-specific mutagenesis methods represented by inverse PCR or CRISPR-Cas9 techniques to increase their fermentation titers in the future.

Integral Stereoselectivity of Lipase Based on the Chromatographic Resolution of Enantiomeric/Regioisomeric Diacylglycerols.

Stereoselectivity, a distinctive characteristic of lipase (EC 3.1.1.3), refers to the ability to differentiate between enantiomeric positions (sn-1 and sn-3) in triacylglycerol (TAG). This property has been determined based on the time course of enantiomeric excess of diacylglycerol (DAG) considering several consecutive steps of lipase-catalyzed hydrolysis of TAG; however, this concept is insufficient to represent the true nature of lipases which are capable of hydrolyzing the sn-2 position of TAG under the condition acyl migration occurs. Here, we suggest "integral stereoselectivity" to capture the preference of lipases for all ester groups of both TAG and DAG, as a novel index of the stereochemistry of lipase. To determine integral stereoselectivity, we established an analytical system based on the chromatographic resolution of dioleoylglycerol (DO) enantiomers and regioisomers. DO enantiomers were derivatized with 4-nitrophenyl isocyanate, and subsequently, resolved by chiral-phase high-performance liquid chromatography-ultraviolet. Regioisomers of monooleoylglycerol and DO were analyzed by HPLC with an evaporative light-scattering detector. Time-course analysis of three model lipases involved in the hydrolysis of trioleoylglycerol validated the analytical system designed to determine the integral stereoselectivity. As an accurate indicator of lipase stereochemistry reflecting all hydrolysis steps, integral stereoselectivity can expedite the development of lipases with unique stereochemistry from agricultural sources and their application to the food industry.

Secretory production in Escherichia coli of a GH46 chitosanase from Chromobacterium violaceum, suitable to generate antifungal chitooligosaccharides.

A chitosanase (CvCsn46) from Chromobacterium violaceum ATCC 12472 was produced in Escherichia coli, purified, and partially characterized. When subjected to denaturing polyacrylamide gel electrophoresis, the enzyme migrated as two protein bands (38 and 36 kDa apparent molecular masses), which were both identified as CvCsn46 by mass spectrometry. The enzyme hydrolyzed colloidal chitosan, with optimum catalytic activity at 50 °C, and two optimum pH values (at pH 6.0 and pH 11.0). The chitosanolytic activity of CvCsn46 was enhanced by some ions (Ca2+, Co2+, Cu2+, Sr2+, Mn2+) and DTT, whereas Fe2+, SDS and ß-mercaptoethanol completely inhibited its activity. CvCsn46 showed a non-Michaelis-Menten kinetics, characterized by a sigmoidal velocity curve (R2 = 0.9927) and a Hill coefficient of 3.95. ESI-MS analysis revealed that the hydrolytic action of CvCsn46 on colloidal chitosan generated a mixture of low molecular mass chitooligosaccharides, containing from 2 to 7 hexose residues, as well as D-glucosamine. The chitosan oligomers generated by CvCsn46 inhibited in vitro the mycelial growth of Lasiodiplodia theobromae, significantly reducing mycelium extension and inducing hyphal morphological alterations, as observed by scanning electron microscopy. CvCsn46 was characterized as a versatile biocatalyst that produces well-defined chitooligosaccharides, which have potential to control fungi that cause important crop diseases.

Whole cell biosynthetic activity of Komagataella phaffii (Pichia pastoris) GS115 strains engineered with transgenes encoding Chromobacterium violaceum ω-transaminase alone or combined with native transketolase.

Whole cell biocatalysis is an ideal tool for biotransformations that demand enzyme regeneration or robustness to fluctuating pH, osmolarity and biocontaminant load in feedstocks. The methylotrophic yeast Komagataella phaffii is an attractive alternative to Escherichia coli for whole cell biocatalysis due to its genetic tractability and capacity to grow to up to 60% wet cell weight by volume. We sought to exploit high cell density K. phaffii to intensify whole-cell chiral amino-alcohol (CAA) biosynthesis. We engineered two novel K. phaffii GS115 strains: one by inserting a Chromobacterium violaceum ω-transaminase CV2025 transgene, for strain PpTAmCV708, and a second strain, PpTAm-TK16, by also inserting the same CV2025 transgene plus a second transgene for a native transketolase. At high cell density, both strains tolerated high substrate concentrations. When fed three low cost substrates, 200 mM glycolaldehyde, 200 mM hydroxypyruvate and 150 mM methylbenzylamine, PpTAm-TK16 whole cells achieved 0.29 g L-1 hr-1 space-time yield of the acetophenone by-product, a 49-fold increase of the highest levels reported for E. coli whole cells harboring the equivalent pathway. When fed only the low-cost substrate, 150 mM methylbenzylamine, strain PpTAmCV708 achieved a 105-fold increase of reported E. coli whole cell biocatalysis performance, with a space-time yield of 0.62 g L-1 hr-1 of the CAA, 2-amino-1,3,4-butanetriol (ABT). The rapid growth and high biomass characteristics of K. phaffii were successfully exploited for production of ABT by whole-cell biocatalysis at higher levels than the previously achieved with E. coli in the presence of the same substrates.

Insight into the dimer dissociation process of the Chromobacterium violaceum (S)-selective amine transaminase.

One of the main factors hampering the implementation in industry of transaminase-based processes for the synthesis of enantiopure amines is their often low storage and operational stability. Our still limited understanding of the inactivation processes undermining the stability of wild-type transaminases represents an obstacle to improving their stability through enzyme engineering. In this paper we present a model describing the inactivation process of the well-characterized (S)-selective amine transaminase from Chromobacterium violaceum. The cornerstone of the model, supported by structural, computational, mutagenesis and biophysical data, is the central role of the catalytic lysine as a conformational switch. Upon breakage of the lysine-PLP Schiff base, the strain associated with the catalytically active lysine conformation is dissipated in a slow relaxation process capable of triggering the known structural rearrangements occurring in the holo-to-apo transition and ultimately promoting dimer dissociation. Due to the occurrence in the literature of similar PLP-dependent inactivation models valid for other non-transaminase enzymes belonging to the same fold-class, the role of the catalytic lysine as conformational switch might extend beyond the transaminase enzyme group and offer new insight to drive future non-trivial engineering strategies.

Folding and stability of recombinant azoreductase enzyme from Chromobacterium violaceum.

Azoreductase from Chromobacterium violaceum was characterized biophysically using experimental and computational tools. The in-silico docking and cross-linking experiments using glutaraldehyde suggest dimeric nature of the enzyme. The enzyme structure was modelled and also studied using circular dichroism (CD) spectroscopy which suggests 40% α- helix, 30% ß- sheet and 30% random coils. In the modelled structure of the azoreductase, the cofactor flavin mononucleotide (FMN) binding energy was -3.8 kJ/mol. The binding of FMN affects the azoreductase-cofactor complex stability. The stability-folding studies indicate that the cofactor, FMN is required for folding, stability and activity. Overall, the data provides interesting insight into stability and biophysical parameters of the azoreductase protein.

Catalytic Ability Improvement of Phenylalanine Hydroxylase from Chromobacterium violaceum by N-Terminal Truncation and Proline Introduction.

Phenylalanine hydroxylase from Chromobacterium violaceum (CvPAH) is a monomeric enzyme that converts phenylalanine to tyrosine. It shares high amino acid identity and similar structure with a subunit of human phenylalanine hydroxylase that is a tetramer, resulting in the latent application in medications. In this study, semirational design was applied to CvPAH to improve the catalytic ability based on molecular dynamics simulation analyses. Four Nterminal truncated variants and one single point variant were constructed and characterized. The D267P variant showed a 2.1-fold increased thermal stability compared to the wild type, but lower specific activity was noted compared with the wild type. The specific activity of all truncated variants was a greater than 25% increase compared to the wild type, and these variants showed similar or slightly decreased thermostability with the exception of the N-Δ9 variant. Notably, the N-Δ9 variant exhibited a 1.2-fold increased specific activity, a 1.3-fold increased thermostability and considerably increased catalytic activity under the neutral environment compared with the wild type. These properties of the N-Δ9 variant could advance medical and pharmaceutical applications of CvPAH. Our findings indicate that the N-terminus might modulate substrate binding, and are directives for further modification and functional research of PAH and other enzymes.

Biochemical characterization and application of a new lipase and its cognate foldase obtained from a metagenomic library derived from fat-contaminated soil.

LipMF3 is a new lipase isolated from a metagenomic library derived from a fat-contaminated soil. It belongs to the lipase subfamily I.1 and has identities of 68% and 67% with lipases of Chromobacterium violaceum and C. amazonense, respectively. Genes encoding LipMF3 and its cognate foldase, LifMF3, were cloned and co-expressed in Escherichia coli. The highest hydrolytic activity of purified Lip-LifMF3 was at 40 °C and pH 6.5. Under these conditions, the highest activity was against tributyrin (1650 U mg-1), but it also had high activity against olive oil (862 U mg-1). It was stable in hydrophilic organic solvents (25%, v/v in water) with residual activity around 100% after 24 h. It also showed stability over a wide pH range (5.5 to 11) with residual activity above 80% after 24 h. Lip-LifMF3 was immobilized by covalent bonding onto Immobead 150P and by adsorption onto Sepabeads FP-BU. The latter preparation gave the best results, producing 94% conversion after 5 h for the synthesis of ethyl oleate and a 90% enantiomeric excess of the product (R)­1­phenylethyl acetate for the kinetic resolution of (R,S)­1­phenyl­1­ethanol. The results obtained in this work provide a basis for the development of applications of Lip-LifMF3 in biocatalysis.

A thermostable chitinase from the antagonistic Chromobacterium violaceum that inhibits the development of phytopathogenic fungi.

The biocontrol activity of some soil strains of Chromobacterium sp. against pathogenic fungi has been attributed to secreted chitinases. The aim of this work was to characterize biochemically a recombinant chitinase (CvChi47) from C. violaceum ATCC 12472 and to investigate its effects on phytopathogenic fungi. CvChi47 is a modular enzyme with 450 amino acid residues, containing a type I signal peptide at the N-terminal region, followed by one catalytic domain belonging to family 18 of the glycoside hydrolases, and two type-3 chitin-binding domains at the C-terminal end. The recombinant enzyme was expressed in Escherichia coli as a His-tagged protein and purified to homogeneity. The native signal peptide of CvChi47 was used to direct its secretion into the culture medium, from where the recombinant product was purified by affinity chromatography on chitin and immobilized metal. The purified protein showed an apparent molecular mass of 46 kDa, as estimated by denaturing polyacrylamide gel electrophoresis, indicating the removal of the signal peptide. CvChi47 was a thermostable protein, retaining approximately 53.7% of its activity when heated at 100 °C for 1 h. The optimum hydrolytic activity was observed at 60 °C and pH 5. The recombinant chitinase inhibited the conidia germination of the phytopathogenic fungi Fusarium oxysporum and F. guttiforme, hence preventing mycelial growth. Furthermore, atomic force microscopy experiments revealed a pronounced morphological alteration of the cell surface of conidia incubated with CvChi47 in comparison to untreated cells. Taken together, these results show the potential of CvChi47 as a molecular tool to control plant diseases caused by these Fusarium species.

Expression optimization, purification, and functional characterization of cholesterol oxidase from Chromobacterium sp. DS1.

Cholesterol oxidase is a bifunctional bacterial flavoenzyme which catalyzes oxidation and isomerization of cholesterol. This valuable enzyme has attracted a great deal of attention because of its wide application in the clinical laboratory, synthesis of steroid derived drugs, food industries, and its potentially insecticidal activity. Therefore, development of an efficient protocol for overproduction of cholesterol oxidase could be valuable and beneficial in this regard. The present study examined the role of various parameters (host strain, culture media, induction time, isopropyl ß-D-1-thiogalactopyranoside concentration, as well as post-induction incubation time and temperature) on over-expression of cholesterol oxidase from Chromobacterium sp. DS1. Applying the optimized protocol, the yield of recombinant cholesterol oxidase significantly increased from 92 U/L to 2115 U/L. Under the optimized conditions, the enzyme was produced on a large-scale, and overexpressed cholesterol oxidase was purified from cell lysate by column nickel affinity chromatography. Km and Vmax values of the purified enzyme for cholesterol were estimated using Lineweaver-Burk plot. Further, the optimum pH and optimum temperature for the enzyme activity were determined. This study reports a straightforward protocol for cholesterol oxidase production which can be performed in any laboratory.

Highly efficient production of chiral amines in batch and continuous flow by immobilized ω-transaminases on controlled porosity glass metal-ion affinity carrier.

In this study, two stereocomplementary ω-transaminases from Arthrobacter sp. (AsR-ωTA) and Chromobacterium violaceum (Cv-ωTA) were immobilized via iron cation affinity binding onto polymer-coated controlled porosity glass beads (EziG™). The immobilization procedure was studied with different types of carrier materials and immobilization buffers of varying compositions, concentrations, pHs and cofactor (PLP) concentrations. Notably, concentrations of PLP above 0.1 mM were correlated with a dramatic decrease of the immobilization yield. The highest catalytic activity, along with quantitative immobilization, was obtained in MOPS buffer (100 mM, pH 8.0, PLP 0.1 mM, incubation time 2 h). Leaching of the immobilized enzyme was not observed within 3 days of incubation. EziG-immobilized AsR-ωTA and Cv-ωTA retained elevated activity when tested for the kinetic resolution of rac-α-methylbenzylamine (rac-α-MBA) in single batch experiments. Recycling studies demonstrated that immobilized EziG3-AsR-ωTA could be recycled for at least 16 consecutive cycles (15 min per cycle) and always affording quantitative conversion (TON ca. 14,400). Finally, the kinetic resolution of rac-α-MBA with EziG3-AsR-ωTA was tested in a continuous flow packed-bed reactor (157 µL reactor volume), which produced more than 5 g of (S)-α-MBA (>49% conversion, >99% ee) in 96 h with no detectable loss of catalytic activity. The calculated TON was more than 110,000 along with a space-time yield of 335 g L-1 h-1.

PHA synthase (PhaC): interpreting the functions of bioplastic-producing enzyme from a structural perspective.

Polyhydroxyalkanoates (PHAs) are biopolymers synthesized by a wide range of bacteria, which serve as a promising candidate in replacing some conventional petrochemical-based plastics. PHA synthase (PhaC) is the key enzyme in the polymerization of PHA, and the crystal structures were successfully determined using the catalytic domain of PhaC from Cupriavidus necator (PhaCCn-CAT) and Chromobacterium sp. USM2 (PhaCCs-CAT). Here, we review the beneficial mutations discovered in PhaCs from a structural perspective. The structural comparison of the residues involved in beneficial mutation reveals that the residues are near to the catalytic triad, but not inside the catalytic pocket. For instance, Ala510 of PhaCCn is near catalytic His508 and may be involved in the open-close regulation, which presumably play an important role in substrate specificity and activity. In the class II PhaC1 from Pseudomonas sp. 61-3 (PhaC1Ps), Ser325 stabilizes the catalytic cysteine through hydrogen bonding. Another residue, Gln508 of PhaC1Ps is located in a conserved hydrophobic pocket which is next to the catalytic Asp and His. A class I, II-conserved Phe420 of PhaCCn is one of the residues involved in dimerization and its mutation to serine greatly reduced the lag phase. The current structural analysis shows that the Phe362 and Phe518 of PhaC from Aeromonas caviae (PhaCAc) are assisting the dimer formation and maintaining the integrity of the core beta-sheet, respectively. The structure-function relationship of PhaCs discussed in this review will serve as valuable reference for future protein engineering works to enhance the performance of PhaCs and to produce novel biopolymers.

Biochemical characterization of a stable azoreductase enzyme from Chromobacterium violaceum: Application in industrial effluent dye degradation.

The presence of dye, including azo functional group (NN) containing dyes, in industrial waste water is one of the major causes of water pollution. This report showcases the functional role of azoreductase from Chromobacterium violaceum (MTCC No: 2656) as a valuable enzyme for degradation of azo dyes. The enzyme was cloned, expressed, purified and biochemically characterized and further tested for degradation efficiency of azo group containing dyes like methyl red, amaranth and methyl orange. The degraded azo dye products (metabolites) resulted by the action of azoreductase enzyme had reduced toxicity on fibroblast cell lines (L929) as compared to raw and intact dye. Further, good stability of the enzyme makes it more suitable for various applications related to the degradation and decolourisation of effluent dyes.

Structure-Based Virtual Screening of LsrK Kinase Inhibitors to Target Quorum Sensing.

In the era of increased antibiotic resistance, targeting enzymes involved in bacterial communication (quorum sensing) represents a new strategy to fight bacterial infections. LsrK is a kinase responsible for the phosphorylation of autoinducer-2, a signaling molecule involved in quorum sensing. Inhibiting LsrK would lead to quorum sensing inactivation and interfere with the pathogenesis. In this study, we built the first LsrK 3D model and performed virtual screening of a locally available database. Selected compounds were tested against LsrK, and the analogue search conducted based on the positive hits led to the identification of low-micromolar LsrK inhibitors. These results prove the utility of the model and provide the first class of LsrK inhibitors to be further optimized as antivirulence agents.

Protein engineering for improving the thermostability of tryptophan oxidase and insights from structural analysis.

l-Tryptophan oxidase, VioA from Chromobacterium violaceum, which has a high substrate specificity for tryptophan, is useful for quantitative assay of tryptophan. However, stability of wild type VioA is not enough for its application in clinical or industrial use. To improve the thermal stability of the enzyme, we developed a VioA (C395A) mutant, with higher stability than wild type VioA. The VioA (C395A) exhibited similar specificity and kinetic parameter for tryptophan to wild type. Conventionally, the quantity of tryptophan is determined by instrumental methods, such as high-performance liquid chromatography (HPLC) after pre-column-derivatization. Using the mutant enzyme, we succeeded in the tryptophan quantification in human plasma samples, to an accuracy of <2.9% when compared to the instrumental method, and to a precision of CV <3.2%. To analyse the improvement in storage stability and substrate specificity, we further determined the crystal structures of VioA (C395A) complexed with FAD, and with FAD and tryptophan at 1.8 Å resolution.

Co-immobilized Whole Cells with ω-Transaminase and Ketoreductase Activities for Continuous-Flow Cascade Reactions.

An improved sol-gel process involving the use of hollow silica microspheres as a supporting additive was applied for the co-immobilization of whole cells of Escherichia coli with Chromobacterium violaceum ω-transaminase activity and Lodderomyces elongisporus with ketoreductase activity. The co-immobilized cells with two different biocatalytic activities could perform a cascade of reactions to convert racemic 4-phenylbutan-2-amine or heptan-2-amine into a nearly equimolar mixture of the corresponding enantiomerically pure R amine and S alcohol even in continuous-flow mode. The novel co-immobilized whole-cell system proved to be an easy-to-store and durable biocatalyst.

Aminopeptidase secreted by Chromobacterium sp. Panama inhibits dengue virus infection by degrading the E protein.

Dengue virus (DENV) is the most prevalent and burdensome arbovirus transmitted by Aedes mosquitoes, against which there is only a limited licensed vaccine and no approved drug treatment. A Chromobacterium species, C. sp. Panama, isolated from the midgut of A. aegypti is able to inhibit DENV replication within the mosquito and in vitro. Here we show that C. sp. Panama mediates its anti-DENV activity through secreted factors that are proteinous in nature. The inhibitory effect occurs prior to virus attachment to cells, and is attributed to a factor that destabilizes the virion by promoting the degradation of the viral envelope protein. Bioassay-guided fractionation, coupled with mass spectrometry, allowed for the identification of a C. sp. Panama-secreted neutral protease and an aminopeptidase that are co-expressed and appear to act synergistically to degrade the viral envelope (E) protein and thus prevent viral attachment and subsequent infection of cells. This is the first study characterizing the anti-DENV activity of a common soil and mosquito-associated bacterium, thereby contributing towards understanding how such bacteria may limit disease transmission, and providing new tools for dengue prevention and therapeutics.

Biosynthesis of 2-aminooctanoic acid and its use to terminally modify a lactoferricin B peptide derivative for improved antimicrobial activity.

Terminal modification of peptides is frequently used to improve their hydrophobicity. While N-terminal modification with fatty acids (lipidation) has been reported previously, C-terminal lipidation is limited as it requires the use of linkers. Here we report the use of a biocatalyst for the production of an unnatural fatty amino acid, (S)-2-aminooctanoic acid (2-AOA) with enantiomeric excess > 98% ee and the subsequent use of 2-AOA to modify and improve the activity of an antimicrobial peptide. A transaminase originating from Chromobacterium violaceum was employed with a conversion efficiency 52-80% depending on the ratio of amino group donor to acceptor. 2-AOA is a fatty acid with amino functionality, which allowed direct C- and N-terminal conjugation respectively to an antimicrobial peptide (AMP) derived from lactoferricin B. The antibacterial activity of the modified peptides was improved by up to 16-fold. Furthermore, minimal inhibitory concentrations (MIC) of C-terminally modified peptide were always lower than N-terminally conjugated peptides. The C-terminally modified peptide exhibited MIC values of 25 µg/ml for Escherichia coli, 50 µg/ml for Bacillus subtilis, 100 µg/ml for Salmonella typhimurium, 200 µg/ml for Pseudomonas aeruginosa and 400 µg/ml for Staphylococcus aureus. The C-terminally modified peptide was the only peptide tested that showed complete inhibition of growth of S. aureus.