scholarly journals Purification, Characterization, and Structural Studies of a Sulfatase from Pedobacter yulinensis

Molecules ◽  
2021 ◽  
Vol 27 (1) ◽  
pp. 87
Author(s):  
Caleb R. Schlachter ◽  
Andrea O’Malley ◽  
Linda L. Grimes ◽  
John J. Tomashek ◽  
Maksymilian Chruszcz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Drugs Of Abuse ◽  
Biochemical Characterization ◽  
Genomic Data ◽  
Organic Substrates ◽  
Structural Studies ◽  
Biotechnological Applications ◽  
Sulfatase Activity

Sulfatases are ubiquitous enzymes that hydrolyze sulfate from sulfated organic substrates such as carbohydrates, steroids, and flavones. These enzymes can be exploited in the field of biotechnology to analyze sulfated metabolites in humans, such as steroids and drugs of abuse. Because genomic data far outstrip biochemical characterization, the analysis of sulfatases from published sequences can lead to the discovery of new and unique activities advantageous for biotechnological applications. We expressed and characterized a putative sulfatase (PyuS) from the bacterium Pedobacter yulinensis. PyuS contains the (C/S)XPXR sulfatase motif, where the Cys or Ser is post-translationally converted into a formylglycine residue (FGly). His-tagged PyuS was co-expressed in Escherichia coli with a formylglycine-generating enzyme (FGE) from Mycobacterium tuberculosis and purified. We obtained several crystal structures of PyuS, and the FGly modification was detected at the active site. The enzyme has sulfatase activity on aromatic sulfated substrates as well as phosphatase activity on some aromatic phosphates; however, PyuS did not have detectable activity on 17α-estradiol sulfate, cortisol 21-sulfate, or boldenone sulfate.

scholarly journals Structural studies on the catalytic mechanism of Diaminopropionate ammonia lyase

2014 ◽  
Vol 70 (a1) ◽  
pp. C1822-C1822
Author(s):  
Geeta Deka ◽  
Shveta Bisht ◽  
H.S. Savithri ◽  
M.R.N Murthy
Keyword(s):  
Reaction Mechanism ◽  
Disulfide Bond ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Catalytic Efficiency ◽  
Peptide Antibiotics ◽  
Type Ii ◽  
Structural Studies ◽  
Fold Type

Diaminopropionate ammonia lyase (DAPAL) is a non-stereo specific fold-type II pyridoxal 5' phosphate (PLP) dependent enzyme that catalyzes the conversion of both D/L isoforms of the nonstandard amino acid Diaminopropionate (DAP) to pyruvate and ammonia. DAP is important for the synthesis of nonribosomal peptide antibiotics such as viomycin and capreomycin. Earlier structural studies on EcDAPAL bound to a reaction intermediate (aminoacrylate) suggested that the enzyme follows a two base mechanism, where Asp120 and Lys77 function as general bases to abstract proton from D-DAP and L-DAP respectively. A novel disulfide was observed near the active site, although its functional significance was not clear. In the present study, structural and biochemical characterization of active site mutants Asp120 (Asp120Asn/Ser/Thr/Cys) and Lys77 (Lys77His/ Thr/Ala/Val) of EcDAPAL has been carried out. Reduction of catalytic efficiency (Kcat/Km) of D120N EcDAPAL for D-DAP by 140 fold and presence of the uncatalyzed ligand at the active site in the crystal structure suggested that Asp120 indeed abstracts proton from D-DAP. Lys77, which was speculated to be important for proton abstraction from L DAP, however seemed to be crucial for PLP binding only. Presence of non-covalently bound PLP in the L77W mutant and occurence of both the ketoenamine, enolimine forms of internal aldimine in L77R mutant provided an in depth insight into the unique chemistry of internal aldimine formation in PLP dependent enzymes. To investigate the role of the novel disulfide bond near the active site, C265 and C291 were mutated to Serine. Studies on these mutants show that this disulfide bond gives additional stability to the protein and might regulate the entry of substrates to the active site. Thus, these studies provide deeper insights into the reaction mechanism of EcDAPAL, representing the overall reaction mechanism followed by several other fold-type II PLP pendent enzymes.

scholarly journals Hydroxo-Bridged Active Site of Flavodiiron NO Reductase Revealed by Spectroscopy and Computations

2020 ◽  
Author(s):  
Filipe Folgosa ◽  
Vladimir Pelmenschikov ◽  
Matthias Keck ◽  
Christian Lorent ◽  
Yoshitaka Yoda ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Density Functional Theory ◽  
Data Collection ◽  
Active Site ◽  
Density Functional ◽  
Nuclear Resonance ◽  
Structural Studies ◽  
Functional Theory ◽  
Nuclear Resonance Vibrational Spectroscopy ◽  
Ligand Interactions

<p>NO and O<sub>2</sub> are detoxified in many organisms using flavodiiron proteins (FDPs). The exact coordination of the iron centre in the active site of these enzymes remains unclear despite numerous structural studies. Here, we used <sup>57</sup>Fe nuclear resonance vibrational spectroscopy (NRVS) to probe the iron-ligand interactions in <i>Escherichia coli</i> FDP. This data combined with density functional theory (DFT) and <sup>57</sup>Fe Mössbauer spectroscopy indicate that the oxidised form of FDP contains a dihydroxo-diferric Fe(III)–(µOH<sup>–</sup>)<sub>2</sub>–Fe(III) active site, while its reduction gives rise to a monohydroxo-diferrous Fe(II)–(µOH<sup>–</sup>)–Fe(II) site upon elimination of one bridging OH<sup>–</sup> ligand, thereby providing an open coordination site for NO binding. Prolonged NRVS data collection of the oxidised FDP resulted in photoreduction and formation of a partially reduced diiron center with two bridging hydroxyl ligands. These results have crucial implications for studying and understanding the mechanism of FDP as well as other non-haem diiron enzymes.</p>

scholarly journals Modulation of Monomer Conformation of the BglG Transcriptional Antiterminator from Escherichia coli

2004 ◽  
Vol 186 (20) ◽  
pp. 6775-6781 ◽  
Author(s):  
Liat Fux ◽  
Anat Nussbaum-Shochat ◽  
Livnat Lopian ◽  
Orna Amster-Choder
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Rna Binding ◽  
Cross Linking ◽  
Structural Studies ◽  
Phosphorylated Proteins ◽  
Close Proximity ◽  
Compact Conformation

ABSTRACT The BglG protein positively regulates expression of the bgl operon in Escherichia coli by binding as a dimer to the bgl transcript and preventing premature termination of transcription in the presence of β-glucosides. BglG activity is negatively controlled by BglF, the β-glucoside phosphotransferase, which reversibly phosphorylates BglG according to β-glucoside availability, thus modulating its dimeric state. BglG consists of an RNA-binding domain and two homologous domains, PRD1 and PRD2. Based on structural studies of a BglG homologue, the two PRDs fold similarly, and the interactions within the dimer are PRD1-PRD1 and PRD2-PRD2. We have recently shown that the affinity between PRD1 and PRD2 of BglG is high, and a fraction of the BglG monomers folds in the cell into a compact conformation, in which PRD1 and PRD2 are in close proximity. We show here that both BglG forms, the compact and noncompact, bind to the active site-containing domain of BglF, IIBbgl, in vitro. The interaction of BglG with IIBbgl or BglF is mediated by PRD2. Both BglG forms are detected as phosphorylated proteins after in vitro phosphorylation with IIBbgl and are dephosphorylated by BglF in vitro in the presence of β-glucosides. Nevertheless, genetic evidence indicates that the interaction of IIBbgl and BglF with the compact form is seemingly less favorable. Using in vivo cross-linking, we show that BglF enhances folding of BglG into a compact conformation, whereas the addition of β-glucosides reduces the amount of this form. Based on these results we suggest a model for the modulation of BglG conformation and activity by BglF.

Structural studies on the active site of Escherichia coli RNA polymerase. 2. Geometrical relationship of the interacting substrates

Biochemistry ◽  
1990 ◽  
Vol 29 (25) ◽  
pp. 5994-6002 ◽  
Author(s):  
Richard B. Beal ◽  
Rajasekharan P. Pillai ◽  
Peter P. Chuknyisky ◽  
Abraham Levy ◽  
Edward Tarien ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Active Site ◽  
Structural Studies ◽  
Relationship Of ◽  
Geometrical Relationship

scholarly journals Biochemical Characterization of the 20S Proteasome from the Methanoarchaeon Methanosarcina thermophila

1998 ◽  
Vol 180 (6) ◽  
pp. 1480-1487 ◽  
Author(s):  
Julie A. Maupin-Furlow ◽  
Henry C. Aldrich ◽  
James G. Ferry
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Biochemical Characterization ◽  
Sodium Dodecyl ◽  
20S Proteasome ◽  
Β Subunit ◽  
Basic Residue ◽  
Methanosarcina Thermophila ◽  
Monovalent Ions

ABSTRACT The 20S proteasome from the methanoarchaeon Methanosarcina thermophila was produced in Escherichia coli and characterized. The biochemical properties revealed novel features of the archaeal 20S proteasome. A fully active 20S proteasome could be assembled in vitro with purified native α ring structures and β prosubunits independently produced in Escherichia coli, which demonstrated that accessory proteins are not essential for processing of the β prosubunits or assembly of the 20S proteasome. A protein complex with a molecular mass intermediate to those of the α7 ring and the 20S proteasome was detected, suggesting that the 20S proteasome is assembled from precursor complexes. The heterologously produced M. thermophila 20S proteasome predominately catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residues Phe and Tyr (chymotrypsinlike activity) in short chromogenic and fluorogenic peptides. Low-level hydrolyzing activities were also detected carboxyl to the acidic residue Asp and the basic residue Arg (trypsinlike activity). Sodium dodecyl sulfate and divalent or monovalent ions stimulated chymotrypsinlike activity and inhibited postglutamyl activity, whereas ATP stimulated postglutamyl activity but had little effect on the chymotrypsinlike activity. The results suggest that the 20S proteasome is a flexible protein which adjusts to binding of substrates. The 20S proteasome also hydrolyzed large proteins. Replacement of the nucleophilic Thr1 residue with an Ala in the β subunit abolished all activities, which suggests that only one active site is responsible for the multisubstrate activity. Replacement of β subunit active-site Lys33 with Arg reduced all activities, which further supports the existence of one catalytic site; however, this result also suggests a role for Lys33 in polarization of the Thr1 N, which serves to strip a proton from the active-site Thr1 Oγ nucleophile. Replacement of Asp51 with Asn had no significant effect on trypsinlike activity, enhanced postglutamyl and trypsinlike activities, and only partially reduced lysozyme-hydrolyzing activity, which suggested that this residue is not essential for multisubstrate activity.

scholarly journals Characterization of a New DyP-Peroxidase from the Alkaliphilic Cellulomonad, Cellulomonas bogoriensis

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1208 ◽  
Author(s):  
Mohamed H. Habib ◽  
Henriëtte J. Rozeboom ◽  
Marco W. Fraaije
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Active Site ◽  
Biochemical Characterization ◽  
Sequence Motif ◽  
Acidic Ph ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Ph Values

DyP-type peroxidases are heme-containing enzymes that have received increasing attention over recent years with regards to their potential as biocatalysts. A novel DyP-type peroxidase (CboDyP) was discovered from the alkaliphilic cellulomonad, Cellulomonas bogoriensis, which could be overexpressed in Escherichia coli. The biochemical characterization of the recombinant enzyme showed that it is a heme-containing enzyme capable to act as a peroxidase on several dyes. With the tested substrates, the enzyme is most active at acidic pH values and is quite tolerant towards solvents. The crystal structure of CboDyP was solved which revealed atomic details of the dimeric heme-containing enzyme. A peculiar feature of CboDyP is the presence of a glutamate in the active site which in most other DyPs is an aspartate, being part of the DyP-typifying sequence motif GXXDG. The E201D CboDyP mutant was prepared and analyzed which revealed that the mutant enzyme shows a significantly higher activity on several dyes when compared with the wild-type enzyme.

scholarly journals Probing the Plasticity in the Active Site of Protein N-terminal Methyltransferase 1 Using Bisubstrate Analogs

2020 ◽  
Author(s):  
Dongxing Chen ◽  
Cheng Dong ◽  
Guangping Dong ◽  
Karthik Srinivasan ◽  
Jinrong Min ◽  
...  
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Biochemical Characterization ◽  
Potent Inhibitor ◽  
Structural Studies ◽  
Cofactor Binding ◽  
Bisubstrate Analogs ◽  
Bisubstrate Inhibitors ◽  
General Guidance ◽  
Methyltransferase 1

AbstractThe interactions of a series of bisubstrate analogs with protein N-terminal methyltransferase 1 (NTMT1) were examined to probe the molecular properties of the NTMT1 active site through biochemical characterization and structural studies. Our results indicate that a 2-C to 4-C atom linker enables its respective bisubstrate analog to occupy both substrate and cofactor binding sites of NTMT1, but the bisubstrate analog with a 5-C atom linker only interacts with the substrate binding site and functions as a substrate. Furthermore, the 4-C atom linker is the optimal and produces the most potent inhibitor (Ki, app = 130 ± 40 pM) for NTMT1 to date, displaying over 100,000-fold selectivity over other methyltransferases and 3,000-fold even to its homolog NTMT2. This study reveals the molecular basis for the plasticity of the NTMT1 active site. Additionally, our study outlines a general guidance on the development of bisubstrate inhibitors for any methyltransferases.

scholarly journals Biochemical Characterization of Mycobacterium tuberculosis DNA Repair Enzymes – Nfo, XthA and Nei2

2014 ◽  
Vol 2 ◽  
Author(s):  
Sailau Abeldenov ◽  
Murat Saparbayev ◽  
Bekbolat Khassenov
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Mycobacterium Tuberculosis ◽  
Recombinant Proteins ◽  
Biochemical Characterization ◽  
Ion Trap ◽  
Affinity Column ◽  
Dna Repair Enzymes ◽  
Repair Enzymes

Introduction: Tuberculosis (TB) is a human disease caused by Mycobacterium tuberculosis (Mtb). Treatment of TB requires long-term courses of multi-drug therapies to eliminate subpopulations of bacteria, which sometimes persist against antibiotics. Therefore, understanding of the mechanism of Mtb antibiotic-resistance is extremely important. During infection, Mtb overcomes a variety of body defense mechanisms, including treatment with the reactive species of oxygen and nitrogen. The bases in DNA molecule are susceptible to the damages caused by reactive forms of intermediate compounds of oxygen and nitrogen. Most of this damage is repaired by the base excision repair (BER) pathway. In this study, we aimed to biochemically characterize three Mtb DNA repair enzymes of BER pathway.Methods: XthA, nfo, and nei genes were identified in mycobacteria by homology search of genomic sequences available in the GenBank database. We used standard methods of genetic engineering  to clone and sequence Mtb genes, which coded Nfo, XthA and Nei2 repair enzymes. The protein products of Mtb genes were expressed and purified in Escherichia coli using affinity tags. The enzymatic activity of purified Nfo, XthA, and Nei2 proteins were measured using radioactively labeled DNA substrates containing various modified residues. Results: The genes end (Rv0670), xthA (Rv0427c), and nei (Rv3297) were PCR amplified using genomic DNA of Mtb H37Rv with primers that contain specific restriction sites. The amplified products were inserted into pET28c(+) expression vector in such a way that the recombinant proteins contain C-terminal histidine tags. The plasmid constructs were verified by sequencing and then transformed into the Escherichia coli BL21 (DE3) strain. Purification of recombinant proteins was performed using Ni2+ ions immobilized affinity column, coupled with the fast performance liquid chromatography machine AKTA. Identification of the isolated proteins was performed by protein mass spectrometry by ion trap tandem MS/MS on nLC-ESI-Ion-Trap platform. Biochemical characterization of DNA repair protein-catalyzed activity was carried out by measuring apurinic/apyrimidinic endonuclease, DNA glycosylase, exonuclease, and 3'-repair diesterase functions. In addition, effect of the opposite base and the influence of metal ion cofactors were measured. Conclusion: Results of the ongoing study will help us define the role of DNA repair enzymes in the emergence of mutations in the mycobacterial genome and, possibly, the origins of multi-drug resistance in mycobacteria.  

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