Dienelactone Hydrolase Research Articles

Unveiling the crystal structure of thermostable dienelactone hydrolase exhibiting activity on terephthalate esters

Dienelactone hydrolase (DLH) is one of numerous hydrolytic enzymes with an α/β-hydrolase fold, which catalyze the hydrolysis of dienelactone to maleylacetate. The DLHs share remarkably similar tertiary structures and a conserved arrangement of catalytic residues. This study presents the crystal structure and comprehensive functional characterization of a novel thermostable DLH from the bacterium Hydrogenobacter thermophilus (HtDLH). The crystal structure of the HtDLH, solved at a resolution of about 1.67 Å, exhibits a canonical α/β-hydrolase fold formed by eight β-sheet strands in the core, with one buried α-helix and six others exposed to the solvent. The structure also confirmed the conserved catalytic triad of DHLs formed by Cys121, Asp170, and His202 residues. The HtDLH forms stable homodimers in solution. Functional studies showed that HtDLH has the expected esterase activity over esters with short carbon chains, such as p-nitrophenyl acetate, reaching optimal activity at pH 7.5 and 70 °C. Furthermore, HtDLH maintains more than 50 % of its activity even after incubation at 90 °C for 16 h. Interestingly, HtDLH exhibits catalytic activity towards polyethylene terephthalate (PET) monomers, including bis-1,2-hydroxyethyl terephthalate (BHET) and 1-(2-hydroxyethyl) 4-methyl terephthalate, as well as other aliphatic and aromatic esters. These findings associated with the lack of activity on amorphous PET indicate that HtDLH has characteristic of a BHET-degrading enzyme. This work expands our understanding of enzyme families involved in PET degradation, providing novel insights for plastic biorecycling through protein engineering, which could lead to eco-friendly solutions to reduce the accumulation of plastic in landfills and natural environments.

Phylum-level studies of bacterial cutinases for unravelling enzymatic specificity toward PET degradation: an in silico approach.

The overwhelming use of PET plastic in various day-to-day activities led to the voluminous increase in PET waste and growing environmental hazards. A plethora of methods have been used that are associated with secondary pollutants. Therefore, microbial degradation of PET provides a sustainable approach due to its versatile metabolic diversity and capacity. The present work highlights the cutinase enzyme's role in PET degradation. This study focuses on the bacterial cutinases homologs screened from 43 reported phylum of bacteria. The reported bacterial cutinases for plastic degradation have been chosen as reference sequences, and 917 sequences have shown homology across the bacterial phyla. The dienelactone hydrolase (DLH) domain was identified for attaining specificity towards PET binding in 196 of 917 sequences. Various computational tools have been used for the physicochemical characterization of 196 sequences. The analysis revealed that most selected sequences are hydrophilic, extracellular, and thermally stable. Based on this analysis, 17 sequences have been further pursued for three-dimensional structure prediction and validation. The molecular docking studies of 17 selected sequences revealed efficient PET binding with the three sequences derived from the phylum Bacteroidota, the lowest binding energy of -5.9kcal/mol, Armatimonadota, and Nitrososphaerota with -5.8kcal/mol. The two enzyme sequences retrieved from the phylum Bacteroidota and Armatimonadota are metagenomically derived. Therefore, the present studies concluded that there is a high probability of finding cutinase homologs in various environmental resources that can be further explored for PET degradation.

Genetic Mechanism of Non-Targeted-Site Resistance to Diquat in Spirodela polyrhiza.

Understanding non-target-site resistance (NTSR) to herbicides represents a pressing challenge as NTSR is widespread in many weeds. Using giant duckweed (Spirodela polyrhiza) as a model, we systematically investigated genetic and molecular mechanisms of diquat resistance, which can only be achieved via NTSR. Quantifying the diquat resistance of 138 genotypes, we revealed an 8.5-fold difference in resistance levels between the most resistant and most susceptible genotypes. Further experiments suggested that diquat uptake and antioxidant-related processes jointly contributed to diquat resistance in S. polyrhiza. Using a genome-wide association approach, we identified several candidate genes, including a homolog of dienelactone hydrolase, that are associated with diquat resistance in S. polyrhiza. Together, these results provide new insights into the mechanisms and evolution of NTSR in plants.

New dienelactone hydrolase from microalgae bacterial community-Antibiofilm activity against fish pathogens and potential applications for aquaculture

Biofilms are resistant to many traditional antibiotics, which has led to search for new antimicrobials from different and unique sources. To harness the potential of aquatic microbial resources, we analyzed the meta-omics datasets of microalgae-bacteria communities and mined them for potential antimicrobial and quorum quenching enzymes. One of the most interesting candidates (Dlh3), a dienelactone hydrolase, is a α/β-protein with predicted eight α-helices and eight β-sheets. When it was applied to one of the major fish pathogens, Edwardsiella anguillarum, the biofilm development was reproducibly inhibited by up to 54.5%. The transcriptome dataset in presence of Dlh3 showed an upregulation in functions related to self-defense like active genes for export mechanisms and transport systems. The most interesting point regarding the biotechnological potential for aquaculture applications of Dlh3 are clear evidence of biofilm inhibition and that health and division of a relevant fish cell model (CHSE-214) was not impaired by the enzyme.

DIENELACTONE HYDROLASE LIKE PROTEIN1 negatively regulates the KAI2-ligand pathway in Marchantia polymorpha

Karrikins are smoke-derived butenolides that induce seed germination and photomorphogenesis in a wide range of plants.1,2,3 KARRIKIN INSENSITIVE2 (KAI2), a paralog of a strigolactone receptor, perceives karrikins or their metabolized products in Arabidopsis thaliana.4,5,6,7 Furthermore, KAI2 is thought to perceive an unidentified plant hormone, called KAI2 ligand (KL).8,9 KL signal is transduced via the interaction between KAI2, MORE AXILLARY GROWTH2 (MAX2), and SUPPRESSOR of MORE AXILLARY GROWTH2 1 LIKE family proteins (SMXLs), followed by the degradation of SMXLs.4,7,10,11,12,13,14 This signaling pathway is conserved both in A.thaliana and the bryophyte Marchantia polymorpha.14 Although the KL signaling pathway is well characterized, the KL metabolism pathways remain poorly understood. Here, we show that DIENELACTONE HYDROLASE LIKE PROTEIN1 (DLP1) is a negative regulator of the KL pathway in M.polymorpha. The KL signal induces DLP1 expression. DLP1 overexpression lines phenocopied the Mpkai2a and Mpmax2 mutants, while dlp1 mutants phenocopied the Mpsmxl mutants. Mutations in the KL signaling genes largely suppressed these phenotypes, indicating that DLP1 acts upstream of the KL signaling pathway, although DLP1 also has KL pathway-independent functions. DLP1 exhibited enzymatic activity toward a potential substrate, suggesting the possibility that DLP1 works through KL inactivation. Investigation of DLP1 homologs in A.thaliana revealed that they do not play a major role in the KL pathway, suggesting different mechanisms for the KL signal regulation. Our findings provide new insights into the regulation of the KL signal in M.polymorpha and the evolution of the KL pathway in land plants.

Enzymatic properties and degradation characterization of a bis(2-hydroxyethyl) terephthalate hydrolase from Saccharothrix sp.

The discovery of new enzymes for poly(ethylene terephthalate) (PET) degradation has been a hot topic of research globally. Bis-(2-hydroxyethyl) terephthalate (BHET) is an intermediate compound in the degradation of PET and competes with PET for the substrate binding site of the PET-degrading enzyme, thereby inhibiting further degradation of PET. Discovery of new BHET degradation enzymes may contribute to improving the degradation efficiency of PET. In this paper, we discovered a hydrolase gene sle (ID: CP064192.1, 5085270-5086049) from Saccharothrix luteola, which can hydrolyze BHET into mono-(2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA). BHET hydrolase (Sle) was heterologously expressed in Escherichia coli using a recombinant plasmid, and the highest protein expression was achieved at a final concentration of 0.4 mmol/L of isopropyl-β-d-thiogalactoside (IPTG), an induction duration of 12 h and an induction temperature of 20 ℃. The recombinant Sle was purified by nickel affinity chromatography, anion exchange chromatography, and gel filtration chromatography, and its enzymatic properties were also characterized. The optimum temperature and pH of Sle were 35 ℃ and 8.0, and more than 80% of the enzyme activity could be maintained in the range of 25-35 ℃ and pH 7.0-9.0 and Co2+ could improve the enzyme activity. Sle belongs to the dienelactone hydrolase (DLH) superfamily and possesses the typical catalytic triad of the family, and the predicted catalytic sites are S129, D175, and H207. Finally, the enzyme was identified as a BHET degrading enzyme by high performance liquid chromatography (HPLC). This study provides a new enzyme resource for the efficient enzymatic degradation of PET plastics.

The Contribution of Actinobacteria to the Degradation of Chlorinated Compounds: Variations in the Activity of Key Degradation Enzymes

Bacteria make a huge contribution to the purification of the environment from toxic stable pollutants of anthropogenic and natural origin due to the diversity of their enzyme systems. For example, the ability to decompose 3-chlorobenzoate (3CBA) by the four representative genera of Actinobacteria, such as Rhodococcus, Gordonia, Microbacterium, and Arthrobacter, was studied. In most cases, the formation of 4-chlorocatechol as the only key intermediate during the decomposition of 3CBA was observed. However, Rhodococcus opacus strain 1CP was an exception, whose cells decomposed 3CBA via both 3-chloro- and 4-chlorocatechol. The enzyme 3-Chlorobenzoate 1,2-dioxygenase (3CBDO) induced during the growth of these bacteria in the presence of 3CBA differed significantly in substrate specificity from the benzoate dioxygenases induced upon growth in the presence of benzoate. The R. opacus 6a strain was found to contain genes encoding chlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, and dienelactone hydrolase, whose nucleotide sequence was 100% consistent with the sequences of the corresponding genes encoding the enzymes of the modified 4-chlorocatechol ortho-cleavage pathway of the strain R. opacus 1CP. However, the gene encoding chloromuconolactone dehalogenase (clcF) was not found in the representatives of the actinomycete genera, including Gordonia and Arthrobacter. A linear mega-plasmid carrying 3-chlorocatechol degradation genes remained stable after maintaining the R. opacus 1CP strain on an agar-rich medium for 25 years. In general, a similar plasmid was absent in actinobacteria of other genera, as well as in closely related species of R. opacus 6a.

Investigating the defoliating‐like (DL) VCG2A pathotype of Verticillium dahliae through identification and prediction of secreted proteins from genomes of Australian isolates

AbstractIn Australia, recent investigations have reported the presence of a number of Verticillium dahliae pathotypes (VCG1A, VCG2A and VCG6) that cause disease in cotton and agricultural weeds. During these investigations, it was observed that a VCG2A V. dahliae displayed greater virulence than previously reported in local and international isolates. Genome sequencing of seven V. dahliae isolates was performed using a mixture of short and long reads sequencing technologies. Of these seven isolates, four were identified as D VCG1A, one ND VCG2A, a recently identified Australian VCG6 as well as a virulent VCG2A, classified as “defoliating‐like”. The secreted protein repertoire from the genomes of these Australian V. dahliae isolates was predicted using four separate signal prediction methods. The consensus secreted protein set of the isolates revealed the presence of 20 proteins that were present in all virulent isolates, including the virulent DL VCG2A, and absent in the nonvirulent VCG2A isolates. Ten of these proteins had a functional annotation and were identified as ligninase H8, lipolytic enzyme, laccase, amine oxidase, spherulin‐1A, dipeptidyl‐peptidase, monooxygenase, dienelactone hydrolase, carboxypeptidase and fibronectin by BLASTP.

Transcriptomic analyses reveal increased expression of dioxygenases, monooxygenases, and other metabolizing enzymes involved in anthracene degradation in the marine alga Ulva lactuca.

To analyze the mechanisms involved in anthracene (ANT) degradation in the marine alga Ulva lactuca, total RNA was obtained from the alga cultivated without ANT and with 5 μM of ANT for 24 h, and transcriptomic analyses were performed. A de novo transcriptome was assembled, transcripts differentially expressed were selected, and those overexpressed were identified. Overexpressed transcripts potentially involved in ANT degradation were: one aromatic ring dioxygenase, three 2-oxoglutarate Fe (II) dioxygenases (2-OGDOs), and three dienelactone hydrolases that may account for anthraquinone, phthalic anhydride, salicylic acid, and phthalic acid production (pathway 1). In addition, two flavin adenine dinucleotide (FAD)-dependent monooxygenases, four cytP450 monooxygenases, two epoxide hydrolase, one hydroxyphenylpyruvic acid dioxygenase (HPPDO), and two homogentisic acid dioxygenases (HGDOs) were identified that may also participate in ANT degradation (pathway 2). Moreover, an alkane monooxygenase (alkB), two alcohol dehydrogenases, and three aldehyde dehydrogenases were identified, which may participate in linear hydrocarbon degradation (pathway 3). Furthermore, the level of transcripts encoding some of mentioned enzymes were quantified by qRT-PCR are in the alga cultivated with 5 μM of ANT for 0–48 h, and those more increased were 2-OGDO, HGDO, and alkB monooxygenase. Thus, at least three pathways for ANT and linear hydrocarbons degradation may be existed in U. lactuca. In addition, ANT metabolites were analyzed by gas chromatography and mass spectrometry (GC–MS), allowing the identification of anthraquinone, phthalic anhydride, salicylic acid, and phthalic acid, thus validating the pathway 1.

In silico approach for identification of polyethylene terephthalate hydrolase (PETase)-like enzymes

Substantial use of plastics as packaging material has become a global concern due to their limited recycling or incineration options and requires periodic degradation to avoid accumulation as a pollutant in the environment. Several chemical and physical properties such as surface area, hydrophobicity, chemical structure, molecular weight, melting temperature, crystallinity, and so on influence their biodegradation. Previously, hydrolases from Thermobifida fusca were reported to have higher degradability until Polyethylene terephthalate hydrolase (PETase) from Ideonella sakaiensis was discovered that showed better specificity for PET. The catalytic triad contains Ser160, Asp206 and His237 along with conserved serine hydrolase motif “Gly-X1-Ser-X2-Gly” having tryptophan at X1 for extending the hydrophobic surface of the active site and cysteine in the vicinity providing thermal stability via additional disulfide bonds. Here, we employed in silico screening techniques for the identification of potential plastic degrading PETases, initiating with sequence similarity search using IsPETase, scrutinizing for the presence of the conserved serine hydrolase motif containing tryptophan at X1 position and cysteine in the vicinity. Five PETases belonging to the diene-lactone hydrolase protein family from different bacterial species were identified in the initial screening, which was further confirmed by molecular docking studies indicating their capacity to bind MHET as substrate for degradation. Our study provides a reliable framework for identification and characterization of PETases involved in biodegradation of plastics which can be further explored for improving their efficiency and suitability under different conditions. This is extremely beneficial approach considering that the increasing demand for microbial enzymes due to the continued accumulation of plastics in the environment.

Revisiting Jatropha curcas Monomeric Esterase: A Dienelactone Hydrolase Compatible with the Electrostatic Catapult Model.

Jatropha curcas contains seeds with a high oil content, suitable for biodiesel production. After oil extraction, the remaining mass can be a rich source of enzymes. However, data from the literature describing physicochemical characteristics for a monomeric esterase from the J. curcas seed did not fit the electrostatic catapult model for esterases/lipases. We decided to reevaluate this J. curcas esterase and extend its characterization to check this apparent discrepancy and gain insights into the enzyme’s potential as a biocatalyst. After anion exchange chromatography and two-dimensional gel electrophoresis, we identified the enzyme as belonging to the dienelactone hydrolase family, characterized by a cysteine as the nucleophile in the catalytic triad. The enzyme displayed a basic optimum hydrolysis pH of 9.0 and an acidic pI range, in contrast to literature data, making it well in line with the electrostatic catapult model. Furthermore, the enzyme showed low hydrolysis activity in an organic solvent-containing medium (isopropanol, acetonitrile, and ethanol), which reverted when recovering in an aqueous reaction mixture. This enzyme can be a valuable tool for hydrolysis reactions of short-chain esters, useful for pharmaceutical intermediates synthesis, due to both its high hydrolytic rate in basic pH and its stability in an organic solvent.

Systematic metadata analysis of brown rot fungi gene expression data reveals the genes involved in Fenton’s reaction and wood decay process

ABSTRACTBrown-rot fungi are rapid holocellulose degraders and are the most predominant degraders of coniferous wood products in North America. Brown-rot fungi degrades wood by both enzymatic (plant biomass degrading carbohydrate active enzymes-CAZymes) and non-enzymatic systems (Fenton’s reaction) mechanisms. Identifying the genes and molecular mechanisms involved in Fenton’s reaction would significantly improve our understanding about brown-rot decay. Our present study identifies the common gene expression patterns involved in brown rot decay by performing metadata analysis of fungal transcriptome datasets. We have also analyzed and compared the genome-wide annotations (InterPro and CAZymes) of the selected brown rot fungi. Genes encoding for various oxidoreductases, iron homeostasis, and metabolic enzymes involved in Fenton’s mechanism were found to be significantly expressed among all the brown-rot fungal datasets. Interestingly, a higher number of hemicellulases encoding genes were differentially expressed among all the datasets, while a fewer number of cellulases and peroxidases were expressed (especially haem peroxidase and chloroperoxidase). Apart from these lignocellulose degrading enzymes genes encoding for aldo/keto reductases, 2-nitro dioxygenase, aromatic-ring dioxygenase, dienelactone hydrolase, alcohol dehydrogenase, major facilitator superfamily, cytochrome-P450 monoxygenase, cytochrome b5, and short-chain dehydrogenase were common and differentially up regulated among all the analyzed brown-rot fungal datasets.

Biochemical and structural characterization of a novel cold-active esterase-like protein from the psychrophilic yeast Glaciozyma antarctica.

Dienelactone hydrolase, an α/β hydrolase enzyme, catalyzes the hydrolysis of dienelactone to maleylacetate, an intermediate for the Krebs cycle. Genome sequencing of the psychrophilic yeast, Glaciozyma antarctica predicted a putative open reading frame (ORF) for dienelactone hydrolase (GaDlh) with 52% sequence similarity to that from Coniophora puteana. Phylogenetic tree analysis showed that GaDlh is closely related to other reported dienelactone hydrolases, and distantly related to other α/β hydrolases. Structural prediction using MODELLER 9.14 showed that GaDlh has the same α/β hydrolase fold as other dienelactone hydrolases and esterase/lipase enzymes, with a catalytic triad consisting of Cys-His-Asp and a G-x-C-x-G-G motif. Based on the predicted structure, GaDlh exhibits several characteristics of cold-adapted proteins such as glycine clustering in the binding pocket, reduced protein core hydrophobicity, and the absence of proline residues in loops. The putative ORF was amplified, cloned, and overexpressed in an Escherichia coli expression system. The recombinant protein was overexpressed as soluble proteins and was purified via Ni-NTA affinity chromatography. Biochemical characterization of GaDlh revealed that it has an optimal temperature at 10°C and that it retained almost 90% of its residual activity when incubated for 90min at 10°C. The optimal pH was at pH 8.0 and it was stable between pH 5-9 when incubated for 60min (more than 50% residual activity). Its Km value was 256μM and its catalytic efficiency was 81.7s-1. To our knowledge, this is the first report describing a novel cold-active dienelactone hydrolase-like protein.

Overexpressed Proteins in Hypervirulent Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 Compared to E. coli O157:H7 EDL933 Clade 3 Strain.

Escherichia coli O157:H7 is responsible for severe diarrhea and hemolytic uremic syndrome (HUS), and predominantly affects children under 5 years. The major virulence traits are Shiga toxins, necessary to develop HUS and the Type III Secretion System (T3SS) through which bacteria translocate effector proteins directly into the host cell. By SNPs typing, E. coli O157:H7 was separated into nine different clades. Clade 8 and clade 6 strains were more frequently associated with severe disease and HUS. In this study, we aimed to identify differentially expressed proteins in two strains of E. coli O157:H7 (clade 8 and clade 6), obtained from cattle and compared them with the well characterized reference EDL933 strain (clade 3). Clade 8 and clade 6 strains show enhanced pathogenicity in a mouse model and virulence-related properties. Proteins were extracted and analyzed using the TMT-6plex labeling strategy associated with two dimensional liquid chromatography and mass spectrometry in tandem. We detected 2241 proteins in the cell extract and 1787 proteins in the culture supernatants. Attention was focused on the proteins related to virulence, overexpressed in clade 6 and 8 strains compared to EDL933 strain. The proteins relevant overexpressed in clade 8 strain were the curli protein CsgC, a transcriptional activator (PchE), phage proteins, Stx2, FlgM and FlgD, a dienelactone hydrolase, CheW and CheY, and the SPATE protease EspP. For clade 6 strain, a high overexpression of phage proteins was detected, mostly from Stx2 encoding phage, including Stx2, flagellin and the protease TagA, EDL933_p0016, dienelactone hydrolase, and Haemolysin A, amongst others with unknown function. Some of these proteins were analyzed by RT-qPCR to corroborate the proteomic data. Clade 6 and clade 8 strains showed enhanced transcription of 10 out of 12 genes compared to EDL933. These results may provide new insights in E. coli O157:H7 mechanisms of pathogenesis.

Compensatory stabilizing role of surface mutations during the directed evolution of dienelactone hydrolase for enhanced activity.

Directed evolution is a common tool employed to generate enzymes suitable for industrial use. High thermal stability is often advantageous or even a requirement for biocatalysts, as such the evolution of protein stability is of practical as well as academic interest. Even when evolving enzymes for new or improved catalytic functions, stability is an important factor since it can limit the accumulation rate and number of desired active site mutations. Dienelactone hydrolase, a small monomeric protein, has been previously evolved via a three-stage process to possess enhanced activity and specificity toward non-physiological substrates. In addition to seven active site mutations there were three surface mutations that were thought to increase the stability of the enzyme and compensate for the destabilizing active site mutations. Here, the individual influence of the three surface mutations--Q110L, Y137C and N154D--on the thermal and chemical stability of DLH has been assessed. While the Q110L and N154D mutations improved the thermal stability, the influence of the Y137C mutation was more complex. Individually it was destabilizing both thermally and chemically, but when in the presence of the Q110L and N154D mutations its effect was neutralized in relation to thermal but not chemical stability. In the context of a directed evolution experiment, these compensatory surface mutations play important roles. However, our results show that detrimental mutations can arise, thus the simultaneous monitoring of stability changes while evolving enzymes for enhanced catalytic properties can be beneficial.

Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search.

The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.

Two structurally different dienelactone hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 plasmid pJP4 catalyse cis- and trans-dienelactones with similar efficiency.

In this study, dienelactone hydrolases (TfdEI and TfdEII) located on plasmid pJP4 of Cupriavidus necator JMP134 were cloned, purified, characterized and three dimensional structures were predicted. tfdEI and tfdEII genes were cloned into pET21b vector and expressed in E. coli BL21(DE3). The enzymes were purified by applying ultra-membrane filtration, anion-exchange QFF and gel-filtration columns. The enzyme activity was determined by using cis-dienelactone. The three-dimensional structure of enzymes was predicted using SWISS-MODEL workspace and the biophysical properties were determined on ExPASy server. Both TfdEI and TfdEII (Mr 25 kDa) exhibited optimum activity at 37°C and pH 7.0. The enzymes retained approximately 50% of their activity after 1 h of incubation at 50°C and showed high stability against denaturing agents. The TfdEI and TfdEII hydrolysed cis-dienelactone at a rate of 0.258 and 0.182 µMs−1, with a Km value of 87 µM and 305 µM, respectively. Also, TfdEI and TfdEII hydrolysed trans-dienelactone at a rate of 0.053 µMs−1 and 0.0766 µMs−1, with a Km value of 84 µM and 178 µM, respectively. The TfdEI and TfdEII kcat/Km ratios were 0.12 µM−1s−1and 0.13 µM−1s−1 and 0.216 µM−1s−1 and 0.094 µM−1s−1 for for cis- and trans-dienelactone, respectively. The kcat/Km ratios for cis-dienelactone show that both enzymes catalyse the reaction with same efficiency even though Km value differs significantly. This is the first report to characterize and compare reaction kinetics of purified TfdEI and TfdEII from Cupriavidus necator JMP134 and may be helpful for further exploration of their catalytic mechanisms.

Crystallization of dienelactone hydrolase in two space groups: structural changes caused by crystal packing.

Dienelactone hydrolase (DLH) is a monomeric protein with a simple α/β-hydrolase fold structure. It readily crystallizes in space group P2₁2₁2₁ from either a phosphate or ammonium sulfate precipitation buffer. Here, the structure of DLH at 1.85 Å resolution crystallized in space group C2 with two molecules in the asymmetric unit is reported. When crystallized in space group P2₁2₁2₁ DLH has either phosphates or sulfates bound to the protein in crucial locations, one of which is located in the active site, preventing substrate/inhibitor binding. Another is located on the surface of the enzyme coordinated by side chains from two different molecules. Crystallization in space group C2 from a sodium citrate buffer results in new crystallographic protein-protein interfaces. The protein backbone is highly similar, but new crystal contacts cause changes in side-chain orientations and in loop positioning. In regions not involved in crystal contacts, there is little change in backbone or side-chain configuration. The flexibility of surface loops and the adaptability of side chains are important factors enabling DLH to adapt and form different crystal lattices.

A poly‐γ‐d‐glutamic acid depolymerase that degrades the protective capsule of Bacillus anthracis

A mixed culture of Pseudomonas fluorescens and Pusillimonas noertemanii, obtained by soil enrichment, elaborated an enzyme (EnvD) which rapidly hydrolysed poly-γ-d-glutamic acid (PDGA), the constituent of the anti-phagocytic capsule conferring virulence on Bacillus anthracis. The EnvD gene is carried on the P. noertemanii genome but co-culture is required for the elaboration of PDGA depolymerase activity. EnvD showed strong sequence homology to dienelactone hydrolases from other Gram-negative bacteria, possessed no general protease activity but cleaved γ-links in both d- and l-glutamic acid-containing polymers. The stability at 37°C was markedly superior to that of CapD, a γ-glutamyltranspeptidase with PDGA depolymerase activity. Recombinant EnvD was recovered from inclusion bodies in soluble form from an Escherichia coli expression vector and the enzyme stripped the PDGA capsule from the surface of B. anthracis Pasteur within 5 min. We conclude from this in vitro study that rEnvD shows promise as a potential therapeutic for the treatment of anthrax.

Substrate‐Induced Conformational Change and Isomerase Activity of Dienelactone Hydrolase and its Site‐Specific Mutants

Studies of the interactions of dienelactone hydrolase (DLH) and its mutants with both E and Z dienelactone substrates show that the enzyme exhibits two different conformational responses specific for hydrolysis of each of its substrate isomers. DLH facilitates hydrolysis of the Z dienelactone through an unusual charge-relay system that is initiated by interaction between the substrate carboxylate and an enzyme arginine residue that activates an otherwise non-nucleophilic cysteine. The E dienelactone does not display this substrate-arginine binding interaction, but instead induces an alternate conformational response that promotes hydrolysis. Furthermore, the substitution of cysteine 123 for serine (C123S) in DLH, instead of inactivating the enzyme as is typical for this active-site mutation, changes the catalysis from substrate hydrolysis to isomerisation. This is due to the deacylation of the acyl-enzyme intermediates being much slower, thereby increasing their lifetimes and allowing for their interconversion through isomerisation, followed by relactonisation.