Characterization of haloacid dehalogenase superfamily acid phosphatase from Staphylococcus lugdunensis.

The haloacid dehalogenase superfamily implicated in bacterial pathogenesis comprises different enzymes having roles in many metabolic pathways. Staphylococcus lugdunensis, a Gram-positive bacterium, is an opportunistic human pathogen causing infections in the central nervous system, urinary tract, bones, peritoneum, systemic conditions and cutaneous infection. The haloacid dehalogenase superfamily proteins play a significant role in the pathogenicity of certain bacteria, facilitating invasion, survival, and proliferation within host cells. The genome of S. lugdunensis encodes more than ten proteins belonging to this superfamily. However, none of them have been characterized. The present work reports the characterization of one of the haloacid dehalogenase superfamily proteins (SLHAD1) from Staphylococcus lugdunensis. The functional analysis revealed that SLHAD1 is a metal-dependent acid phosphatase, which catalyzes the dephosphorylation of phosphorylated metabolites of cellular pathways, including glycolysis, gluconeogenesis, nucleotides, and thiamine metabolism. Based on the substrate specificity and genomic analysis, the physiological function of SLHAD1 in thiamine metabolism has been tentatively assigned. The crystal structure of SLHAD1, lacking 49 residues at the C-terminal, was determined at 1.7 Å resolution with a homodimer in the asymmetric unit. It was observed that SLHAD1 exhibited time-dependent cleavage at a specific point, occurring through a self-initiated process. A combination of bioinformatics, biochemical, biophysical, and structural studies explored unique features of SLHAD1. Overall, the study revealed a detailed characterization of a critical enzyme of the human pathogen Staphylococcus lugdunensis, associated with several life-threatening infections.

Sulisobenzone is a potent inhibitor of the global transcription factor Cra.

Transcription is carried out by the RNA polymerase and is regulated through a series of interactions with transcription factors. Catabolite activator repressor (Cra), a LacI family transcription factor regulates the virulence gene expression in Enterohaemorrhagic Escherichia coli (EHEC) and thus is a promising drug target for the discovery of antivirulence molecules. Here, we report the crystal structure of the effector molecule binding domain of Cra from E. coli (EcCra) in complex with HEPES molecule. Based on the EcCra-HEPES complex structure, ligand screening was performed that identified sulisobenzone as an potential inhibitor of EcCra. The electrophoretic mobility shift assay (EMSA) and in vitro transcription assay validated the sulisobenzone binding to EcCra. Moreover, the isothermal titration calorimetry (ITC) experiments demonstrated a 40-fold higher binding affinity of sulisobenzone (KD 360 nM) compared to the HEPES molecule. Finally, the sulisobenzone bound EcCra complex crystal structure was determined to elucidate the binding mechanism of sulisobenzone to the effector binding pocket of EcCra. Together, this study suggests that sulisobenzone may be a promising candidate that can be studied and developed as an effective antivirulence agent against EHEC.

Crystal structure and molecular characterization of NADP+-farnesol dehydrogenase from cotton bollworm, Helicoverpaarmigera.

Farnesol dehydrogenase (FDL) orchestrates the oxidation reaction catalyzing farnesol to farnesal, a key step in the juvenile hormone (JH) biosynthesis pathway of insects and hence, represents a lucrative target for developing insect growth regulators (IGRs). However, information on the structural and functional characterization of JH-specific farnesol dehydrogenase in insects remains elusive. Herein, we identified a transcript that encodes farnesol dehydrogenase (HaFDL) from Helicoverpa armigera, a major pest of cotton. The investigations of molecular assembly, biochemical analysis and spatio-temporal expression profiling showed that HaFDL exists as a soluble homo-tetrameric form, exhibits a broad substrate affinity and is involved in the JH-specific farnesol oxidation in H. armigera. Additionally, the study presents the first crystal structure of the HaFDL-NADP enzyme complex determined at 1.6 Å resolution. Structural analysis revealed that HaFDL belongs to the NADP-specific cP2 subfamily of the classical short-chain dehydrogenase/reductase (SDR) family and exhibits typical structural features of those enzymes including the conserved nucleotide-binding Rossman-fold. The isothermal titration calorimetry (ITC) showed a high binding affinity (dissociation constant, Kd, 3.43 µM) of NADP to the enzyme. Comparative structural analysis showed a distinct substrate-binding pocket (SBP) loop with a spacious and hydrophobic substrate-binding pocket in HaFDL, consistent with the biochemically observed promiscuous substrate specificity. Finally, based on the crystal structure, substrate modeling and structural comparison with homologs, a two-step reaction mechanism is proposed. Overall, the findings significantly impact and contribute to our understanding of farnesol dehydrogenase functional properties in JH biosynthesis in H. armigera.

Conformational flexibility enables catalysis of phthalate cis-4,5-dihydrodiol dehydrogenase.

Phthalate cis-4,5-dihydrodiol dehydrogenase (PhtC), the second enzyme of the phthalate catabolic pathway, catalyzes the dehydrogenation of cis-4,5-dihydrodiol phthalate (DDP). Here, we report the structural and biochemical characterization of PhtC from Comamonas testosteroni KF1 (PhtCKF1). With biochemical experiments, we have determined the enzyme's catalytic efficiency (kcat/Km) with DDP as 2.6 ± 0.5 M-1s-1, over 10-fold higher than with cis-3,4-dihydrodiol phthalate (CDP). To understand the structural basis of these reactions, the crystal structures of PhtCKF1 in apo-form, the binary complex with NAD+, and the ternary complex with NAD+ and 3-hydroxybenzoate (3HB) were determined. These crystal structures reveal that the binding of 3HB induces a conformational change in the substrate-binding loop. This conformational change causes the opening of the NAD + binding site while trapping the 3HB. The PhtCKF1 crystal structures show that the catalytic domain of PhtCKF1 is larger than that of other structurally characterized homologs and does not align with other cis-diol dehydrogenases. Structural and mutational analysis of the substrate-binding loop residues, Arg164 and Glu167 establish that conformational flexibility of this loop is necessary for positioning the substrate in a catalytically competent pose, as substitution of either of these residues to Ala did not yield the dehydrogenation activity. Further, based on the crystal structures of PhtCKF1 and related structural homologs, a reaction mechanism is proposed. Finally, with the biochemical analysis of a variant M251LPhtCKF1, the broader substrate specificity of this enzyme is explained.

Structural Insights into Dihydroxylation of Terephthalate, a Product of Polyethylene Terephthalate Degradation.

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis-diol, is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDOKF1). TPDOKF1 exhibited substrate specificity for TPA (kcat/Km = 57 ± 9 mM-1 s-1). The TPDOKF1 structure harbors characteristic RO features as well as a unique catalytic domain that rationalizes the enzyme's function. The docking and mutagenesis studies reveal that its substrate specificity for TPA is mediated by the Arg309 and Arg390 residues, positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, as its mutation to alanine decreases the activity (kcat) by 80%. This study delineates the structural features that dictate the substrate recognition and specificity of TPDO. IMPORTANCE Global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential for tackling this. Microbial utilization of this released product, TPA, is an emerging and promising strategy for waste-to-value creation. Research in the last decade has identified terephthalate dioxygenase (TPDO) as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

MD simulation and MM/PBSA identifies phytochemicals as bifunctional inhibitors of SARS-CoV-2.

The global spread of SARS-CoV-2 has resulted in millions of fatalities worldwide, making it crucial to identify potent antiviral therapeutics to combat this virus. We employed structure-assisted virtual screening to identify phytochemicals that can target the two proteases which are essential for SARS-CoV-2 replication and transcription, the main protease and papain-like protease. Using virtual screening and molecular dynamics, we discovered new phytochemicals with inhibitory activity against the two proteases. Isoginkgetin, kaempferol-3-robinobioside, methyl amentoflavone, bianthraquinone, podocarpusflavone A, and albanin F were shown to have the best affinity and inhibitory potential among the compounds, and can be explored clinically for use as inhibitors of novel coronavirus SARS-CoV-2.Communicated by Ramaswamy H. Sarma.

Molecular insights into substrate recognition and catalysis by phthalate dioxygenase from Comamonas testosteroni.

Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDOKF1 from Comamonas testosteroni KF1 and found that it had an apparent kcat/Km for phthalate of 0.58 ± 0.09 µM-1s-1, over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 Å resolution revealed that it is a hexamer comprising two stacked α3 trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDOKF1 larger than that of other characterized ROs. Complexes of PDOKF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.

Deciphering the enigma of missing DNA binding domain of LacI family transcription factors.

Catabolite repressor activator (Cra) is a member of the LacI family transcriptional regulator distributed across a wide range of bacteria and regulates the carbon metabolism and virulence gene expression. In numerous studies to crystallize the apo form of the LacI family transcription factor, the N-terminal domain (NTD), which functions as a DNA-binding domain, has been enigmatically missing from the final resolved structures. It was speculated that the NTD is disordered or unstable and gets cleaved during crystallization. Here, we have determined the crystal structure of Cra from Escherichia coli (EcCra). The structure revealed a well-defined electron density for the C-terminal domain (CTD). However, electron density was missing for the first 56 amino acids (NTD). Our data reveal for the first time that EcCra undergoes a spontaneous cleavage at the conserved Asn 50 (N50) site, which separates the N-terminal DNA binding domain from the C-terminal effector molecule binding domain. With the site-directed mutagenesis, we confirm the involvement of residue N50 in the spontaneous cleavage phenomenon. Furthermore, the Isothermal titration calorimetry (ITC) assay of the EcCra-NTD with DNA showed EcCra-NTD is in a functional conformation state and retains its DNA binding activity.

Biophysical and In-Silico Studies of Phytochemicals Targeting Chorismate Synthase from Drug-Resistant Moraxella Catarrhalis.

Chorismate serves as a crucial precursor for the synthesis of many aromatic compounds essential for the survival and virulence in various bacteria and protozoans. Chorismate synthase, a vital enzyme in the shikimate pathway, is responsible for the formation of chorismate from enolpyruvylshikimate-3-phosphate (EPSP). Moraxella catarrhalis is reported to be resistant to many beta-lactam antibiotics and causes chronic ailments such as otitis media, sinusitis, laryngitis, and bronchopulmonary infections. Here, we have cloned the aroC gene from Moraxella catarrhalis in pET28c and heterologously produced the chorismate synthase (~ 43 kDa) in Escherichia coli BL21(DE3) cells. We have predicted the three-dimensional structure of this enzyme and used the refined model for ligand-based virtual screening against Supernatural Database using PyRx tool that led to the identification of the top three molecules (caffeic acid, gallic acid, and o-coumaric acid). The resultant protein-ligand complex structures were subjected to 50 ns molecular dynamics (MD) simulation using GROMACS. Further, the binding energy was calculated by MM/PBSA approach using the trajectory obtained from MD simulation. The binding affinities of these compounds were validated with ITC experiments, which suggest that gallic acid has the highest binding affinity amongst these three phytochemicals. Together, these results pave the way for the use of these phytochemicals as potential anti-bacterial compounds.

Characterization of dye-decolorizing peroxidase from Bacillus subtilis.

The dye-decolorizing peroxidases (DyPs) belong to a unique heme peroxidase family for their biotechnological potential to detoxify synthetic dyes. In this work, we have biochemically and structurally characterized the dye-decolorizing peroxidase from Bacillus subtilis (BsDyP). The biochemical studies of BsDyP demonstrate that pH 4.0 is optimum for the oxidation of malachite green (MG) and methyl violet (MV). However, it oxidizes the MG with higher catalytic efficiency (kcat/Km = 6.3 × 102 M-1s-1), than MV (kcat/Km = 5.0 × 102 M-1s-1). While reactive black 5 (RB5) is oxidized at pH 3.0 with the catalytic efficiency of kcat/Km = 3.6 × 102 M-1s-1. The calculated thermodynamic parameters by isothermal titration calorimetry (ITC) reveal the feasibility and spontaneity of dyes binding with BsDyP. Further, the crystal structures of a HEPES bound and unbound of BsDyP provide insight into the probable binding sites of the substrates. In BsDyP-HEPES bound structure, the HEPES-1 molecule is found in the heme cavity at the γ-edge, and another HEPES-2 molecule is bound ~16 Å away from the heme that is fenced by Ile231, Arg234, Ser235, Asp239, Glu334, and surface-exposed Tyr335 residues. Furthermore, the molecular docking, simulation, and MMPBSA studies support the binding of dyes at both the sites of BsDyP and produce lower-energy stable BsDyP-dyes complexes. Here, the BsDyP study allows the identification of its two potential binding sites and shows the oxidation of a variety of dyes. Structural and functional insight of BsDyP will facilitate its engineering for the improved decolorization of dyes.

Engineering Burkholderia xenovorans LB400 BphA through Site-Directed Mutagenesis at Position 283.

Biphenyl dioxygenase (BPDO), which is a Rieske-type oxygenase (RO), catalyzes the initial dioxygenation of biphenyl and some polychlorinated biphenyls (PCBs). In order to enhance the degradation ability of BPDO in terms of a broader substrate range, the BphAES283M, BphAEp4-S283M, and BphAERR41-S283M variants were created from the parent enzymes BphAELB400, BphAEp4, and BphAERR41, respectively, by a substitution at one residue, Ser283Met. The results of steady-state kinetic parameters show that for biphenyl, the kcat/Km values of BphAES283M, BphAEp4-S283M, and BphAERR41-S283M were significantly increased compared to those of their parent enzymes. Meanwhile, we determined the steady-state kinetics of BphAEs toward highly chlorinated biphenyls. The results suggested that the Ser283Met substitution enhanced the catalytic activity of BphAEs toward 2,3',4,4'-tetrachlorobiphenyl (2,3',4,4'-CB), 2,2',6,6'-tetrachlorobiphenyl (2,2',6,6'-CB), and 2,3',4,4',5-pentachlorobiphenyl (2,3',4,4',5-CB). We compared the catalytic reactions of BphAELB400 and its variants toward 2,2'-dichlorobiphenyl (2,2'-CB), 2,5-dichlorobiphenyl (2,5-CB), and 2,6-dichlorobiphenyl (2,6-CB). The biochemical data indicate that the Ser283Met substitution alters the orientation of the substrate inside the catalytic site and, thereby, its site of hydroxylation, and this was confirmed by docking experiments. We also assessed the substrate ranges of BphAELB400 and its variants with degradation activity. BphAES283M and BphAEp4-S283M were clearly improved in oxidizing some of the 3-6-chlorinated biphenyls, which are generally very poorly oxidized by most dioxygenases. Collectively, the present work showed a significant effect of mutation Ser283Met on substrate specificity/regiospecificity in BPDO. These will certainly be meaningful elements for understanding the effect of the residue corresponding to position 283 in other Rieske oxygenase enzymes.IMPORTANCE The segment from positions 280 to 283 in BphAEs is located at the entrance of the catalytic pocket, and it shows variation in conformation. In previous works, results have suggested but never proved that residue Ser283 of BphAELB400 might play a role in substrate specificity. In the present paper, we found that the Ser283Met substitution significantly increased the specificity of the reaction of BphAE toward biphenyl, 2,3',4,4'-CB, 2,2',6,6'-CB, and 2,3',4,4',5-CB. Meanwhile, the Ser283Met substitution altered the regiospecificity of BphAE toward 2,2'-dichlorobiphenyl and 2,6-dichlorobiphenyl. Additionally, this substitution extended the range of PCBs metabolized by the mutated BphAE. BphAES283M and BphAEp4-S283M were clearly improved in oxidizing some of the more highly chlorinated biphenyls (3 to 6 chlorines), which are generally very poorly oxidized by most dioxygenases. We used modeled and docked enzymes to identify some of the structural features that explain the new properties of the mutant enzymes. Altogether, the results of this study provide better insights into the mechanisms by which BPDO evolves to change and/or expand its substrate range and its regiospecificity.

Biodegradation of phthalic acid esters (PAEs) and in silico structural characterization of mono-2-ethylhexyl phthalate (MEHP) hydrolase on the basis of close structural homolog.

Three bacterial strains capable of degrading phthalates namely Pseudomonas sp. PKDM2, Pseudomonas sp. PKDE1 and Pseudomonas sp. PKDE2 were isolated and characterized for their degradative potential. These strains efficiently degraded 77.4%-84.4% of DMP, 75.0%-75.7% of DEP and 71.7%-74.7% of DEHP, initial amount of each phthalate is 500mgL-1 of each phthalate, after 44h of incubation. GC-MS results reveal the tentative DEHP degradation pathway, where hydrolases mediate the breakdown of DEHP to phthalic acid (PA) via an intermediate MEHP. MEHP hydrolase is a serine hydrolase which is involved in the reduction of the MEHP to PA. The predicted 3D model of MEHP hydrolase from Pseudomonas mosselii was docked with phthalate monoesters (PMEs) such as MEHP, mono-n-hexyl phthalate (MHP), mono-n-butyl phthalate (MBP) and mono-n-ethyl phthalate (MEP), respectively. Docking results show the distance between the carbonyl carbon of respective phthalate monoester and the hydroxyl group of catalytic serine lies in the range of 2.9 to 3.3Å, which is similar to the ES complex of other serine hydrolases. This structural study highlights the interaction and the role of catalytic residues of MEHP hydrolase involved in the biodegradation of PMEs to phthalate.