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Journal of Bacteriology Feb 2001Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and...
Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)(3). To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins.
Topics: Antibodies, Bacterial; Apoenzymes; Bacterial Proteins; Carrier Proteins; Cloning, Molecular; DNA Primers; Gene Expression Regulation, Bacterial; Models, Chemical; Phosphate-Binding Proteins; Polymerase Chain Reaction; Precipitin Tests; Protein Binding; Proteus mirabilis; Recombinant Fusion Proteins; Repressor Proteins; Sequence Analysis, DNA; Serine Endopeptidases; Two-Hybrid System Techniques; Urease
PubMed: 11157956
DOI: 10.1128/JB.183.4.1423-1433.2001 -
Biochimica Et Biophysica Acta Jun 1998The ThDP dependent enzyme transketolase is a convenient model system to study enzymatic thiamin catalysis. Crystallographic studies of the enzyme have identified the... (Review)
Review
The ThDP dependent enzyme transketolase is a convenient model system to study enzymatic thiamin catalysis. Crystallographic studies of the enzyme have identified the ThDP binding fold, the V-conformation of ThDP as the relevant conformation in enzymatic catalysis and details of enzyme-substrate interactions. Based on this structural information, the function of various active site residues in substrate binding and catalysis has been probed by site-directed mutagenesis.
Topics: Apoenzymes; Crystallography, X-Ray; Enzyme Activation; Models, Molecular; Mutation; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Thiamine; Thiamine Pyrophosphate; Transketolase
PubMed: 9655943
DOI: 10.1016/s0167-4838(98)00082-x -
Scientific Reports Nov 2015Enzymes play a vital role in catalysing almost all chemical reactions that occur in biological systems. Some enzymes must form complexes with non-protein molecules...
Enzymes play a vital role in catalysing almost all chemical reactions that occur in biological systems. Some enzymes must form complexes with non-protein molecules called cofactors to express catalytic activities. Although the control of catalytic reactions via apoenzyme-cofactor complexes has attracted significant attention, the reports have been limited to the microscale. Here, we report a system to express catalytic activity by adhesion of an apoenzyme gel and a cofactor gel. The apoenzyme and cofactor gels act as catalysts when they form a gel assembly, but they lose catalytic ability upon manual dissociation. We successfully construct a system with switchable catalytic activity via adhesion and separation of the apoenzyme gel with the cofactor gel. We expect that this methodology can be applied to regulate the functional activities of enzymes that bear cofactors in their active sites, such as the oxygen transport of haemoglobin or myoglobin and the electron transport of cytochromes.
Topics: Apoenzymes; Catalysis; Catalytic Domain; Cytochromes; Gels; Organic Chemicals; Oxygen
PubMed: 26537172
DOI: 10.1038/srep16254 -
Infection and Immunity Jul 1992Helicobacter pylori, a gram-negative, microaerophilic, spiral-shaped bacterium, is an etiologic agent of human gastritis and peptic ulceration and is highly restricted... (Comparative Study)
Comparative Study
Helicobacter pylori, a gram-negative, microaerophilic, spiral-shaped bacterium, is an etiologic agent of human gastritis and peptic ulceration and is highly restricted to the gastric mucosa of humans. Urease, synthesized at up to 6% of the soluble cell protein, hydrolyzes urea, thereby releasing ammonia, which may neutralize acid, allowing survival of the bacterium and initial colonization of the gastric mucosa. The urease protein is encoded by two subunit genes, ureA and ureB; however, accessory genes are necessary for enzyme activity. H. pylori urease genes were isolated from a cosmid gene bank and subcloned on a 5.8-kb Sau3A partial fragment carrying ureCDAB, corresponding to four open reading frames described by A. Labigne, V. Cussac, and P. Courcoux (J. Bacteriol. 173:1920-1931, 1991). Clones were confirmed as ureas gene sequences by polymerase chain reaction amplification. The recombinant enzyme was purified from the soluble protein of French press lysates of Escherichia coli DH5 alpha(pHP402) by chromatography on DEAE-Sepharose, Phenyl-Sepharose, Mono-Q, and Superose 6 resins. Fractions containing a catalytically inactive apoenzyme were identified by an enzyme-linked immunosorbent assay (ELISA) by using antisera to native UreA (29.5 kDa) and UreB (66 kDa). Purified recombinant urease was indistinguishable from native enzyme on a Superose 6 column and on Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels. The protein reacted specifically on Western blots (immunoblots) with anti-UreA and anti-UreB antibodies and was recognized with an intensity equal to that of the native enzyme in an ELISA using human sera. Clones containing only ureA and ureB also produced an assembled but inactive enzyme. Enzyme activity was not restored by in trans complementation with cloned urease accessory gene sequences from Proteus mirabilis or Morganella morganii. H. pylori urease genes (ureCDAB) subcloned into pACYC184 were also not complemented with any of 1,000 cosmid clones containing H. pylori chromosomal sequences. However, larger clones containing 4.5 kb of DNA downstream of ureB synthesized catalytically active urease when grown in minimal medium. These data indicate that the ureA and ureB genes encoding H. pylori urease are transcribed and translated in E. coli and that these genes alone are sufficient for the synthesis and assembly of the native size enzyme. Genes downstream of ureB, however, are necessary for production of a catalytically active urease.
Topics: Apoenzymes; Base Sequence; Blotting, Southern; Blotting, Western; Cloning, Molecular; DNA; Enzyme-Linked Immunosorbent Assay; Helicobacter Infections; Helicobacter pylori; Molecular Sequence Data; Mutagenesis; Oligonucleotide Probes; Polymerase Chain Reaction; Recombinant Proteins; Restriction Mapping; Urease
PubMed: 1612735
DOI: 10.1128/iai.60.7.2657-2666.1992 -
Protein Science : a Publication of the... Sep 2004Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in prokaryotic coenzyme A (CoA) biosynthesis, directing the transfer of an adenylyl group... (Comparative Study)
Comparative Study
Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in prokaryotic coenzyme A (CoA) biosynthesis, directing the transfer of an adenylyl group from ATP to 4'-phosphopantetheine (Ppant) to yield dephospho-CoA (dPCoA). The crystal structures of Escherichia coli PPAT bound to its substrates, product, and inhibitor revealed an allosteric hexameric enzyme with half-of-sites reactivity, and established an in-line displacement catalytic mechanism. To provide insight into the mechanism of ligand binding we solved the apoenzyme (Apo) crystal structure of PPAT from Mycobacterium tuberculosis. In its Apo form, PPAT is a symmetric hexamer with an open solvent channel. However, ligand binding provokes asymmetry and alters the structure of the solvent channel, so that ligand binding becomes restricted to one trimer.
Topics: Apoenzymes; Binding Sites; Coenzyme A; Crystallography, X-Ray; Escherichia coli Proteins; Ligands; Models, Molecular; Mycobacterium tuberculosis; Nucleotidyltransferases; Protein Conformation; Structural Homology, Protein; Substrate Specificity
PubMed: 15322293
DOI: 10.1110/ps.04816904 -
The Journal of Biological Chemistry Mar 2002We report here the x-ray crystal structure of a soluble catalytically active fragment of the Escherichia coli type I signal peptidase (SPase-(Delta2-75)) in the absence...
We report here the x-ray crystal structure of a soluble catalytically active fragment of the Escherichia coli type I signal peptidase (SPase-(Delta2-75)) in the absence of inhibitor or substrate (apoenzyme). The structure was solved by molecular replacement and refined to 2.4 A resolution in a different space group (P4(1)2(1)2) from that of the previously published acyl-enzyme inhibitor-bound structure (P2(1)2(1)2) (Paetzel, M., Dalbey, R.E., and Strynadka, N.C.J. (1998) Nature 396, 186-190). A comparison with the acyl-enzyme structure shows significant side-chain and main-chain differences in the binding site and active site regions, which result in a smaller S1 binding pocket in the apoenzyme. The apoenzyme structure is consistent with SPase utilizing an unusual oxyanion hole containing one side-chain hydroxyl hydrogen (Ser-88 OgammaH) and one main-chain amide hydrogen (Ser-90 NH). Analysis of the apoenzyme active site reveals a potential deacylating water that was displaced by the inhibitor. It has been proposed that SPase utilizes a Ser-Lys dyad mechanism in the cleavage reaction. A similar mechanism has been proposed for the LexA family of proteases. A structural comparison of SPase and members of the LexA family of proteases reveals a difference in the side-chain orientation for the general base lysine, both of which are stabilized by an adjacent hydroxyl group. To gain insight into how signal peptidase recognizes its substrates, we have modeled a signal peptide into the binding site of SPase. The model is built based on the recently solved crystal structure of the analogous enzyme LexA (Luo, Y., Pfuetzner, R. A., Mosimann, S., Paetzel, M., Frey, E. A., Cherney, M., Kim, B., Little, J. W., and Strynadka, N. C. J. (2001) Cell 106, 1-10) with its bound cleavage site region.
Topics: Apoenzymes; Bacterial Proteins; Binding Sites; Crystallization; Escherichia coli; Lysine; Membrane Proteins; Protein Sorting Signals; Serine; Serine Endopeptidases
PubMed: 11741964
DOI: 10.1074/jbc.M110983200 -
Biochimica Et Biophysica Acta Dec 2000Thermostable glucose isomerases are desirable for production of 55% fructose syrups at >90 degrees C. Current commercial enzymes operate only at 60 degrees C to produce... (Comparative Study)
Comparative Study Review
Thermostable glucose isomerases are desirable for production of 55% fructose syrups at >90 degrees C. Current commercial enzymes operate only at 60 degrees C to produce 45% fructose syrups. Protein engineering to construct more stable enzymes has so far been relatively unsuccessful, so this review focuses on elucidation of the thermal inactivation pathway as a future guide. The primary and tertiary structures of 11 Class 1 and 20 Class 2 enzymes are compared. Within each class the structures are almost identical and sequence differences are few. Structural differences between Class 1 and Class 2 are less than previously surmised. The thermostabilities of Class 1 enzymes are essentially identical, in contrast to previous reports, but in Class 2 they vary widely. In each class, thermal inactivation proceeds via the tetrameric apoenzyme, so metal ion affinity dominates thermostability. In Class 1 enzymes, subunit dissociation is not involved, but there is an irreversible conformational change in the apoenzyme leading to a more thermostable inactive tetramer. This may be linked to reversible conformational changes in the apoenzyme at alkaline pH arising from electrostatic repulsions in the active site, which break a buried Arg-30-Asp-299 salt bridge and bring Arg-30 to the surface. There is a different salt bridge in Class 2 enzymes, which might explain their varying thermostability. Previous protein engineering results are reviewed in light of these insights.
Topics: Aldose-Ketose Isomerases; Amino Acid Sequence; Apoenzymes; Archaea; Arthrobacter; Binding Sites; Catalysis; Cations, Divalent; Disulfides; Enzyme Stability; Hot Temperature; Metals; Models, Molecular; Molecular Sequence Data; Mutation; Protein Conformation; Protein Denaturation; Protein Engineering; Substrate Specificity; Subtilisin; Thermolysin
PubMed: 11150612
DOI: 10.1016/s0167-4838(00)00246-6 -
Journal of Molecular Biology May 2006The iron-sulphur cluster-free hydrogenase (Hmd, EC 1.12.98.2) from methanogenic archaea is a novel type of hydrogenase that tightly binds an iron-containing cofactor....
The iron-sulphur cluster-free hydrogenase (Hmd, EC 1.12.98.2) from methanogenic archaea is a novel type of hydrogenase that tightly binds an iron-containing cofactor. The iron is coordinated by two CO molecules, one sulphur and a pyridone derivative, which is linked via a phosphodiester bond to a guanosine base. We report here on the crystal structure of the Hmd apoenzyme from Methanocaldococcus jannaschii at 1.75 A and from Methanopyrus kandleri at 2.4 A resolution. Homodimeric Hmd reveals a unique architecture composed of one central and two identical peripheral globular units. The central unit is composed of the intertwined C-terminal segments of both subunits, forming a novel intersubunit fold. The two peripheral units consist of the N-terminal domain of each subunit. The Rossmann fold-like structure of the N-terminal domain contains a mononucleotide-binding site, which could harbour the GMP moiety of the cofactor. Another binding site for the iron-containing cofactor is most probably Cys176, which is located at the bottom of a deep intersubunit cleft and which has been shown to be essential for enzyme activity. Adjacent to the iron of the cofactor modelled as a ligand to Cys176, an extended U-shaped extra electron density, interpreted as a polyethyleneglycol fragment, suggests a binding site for the substrate methenyltetrahydromethanopterin.
Topics: Amino Acid Sequence; Apoenzymes; Binding Sites; Conserved Sequence; Crystallography, X-Ray; Dimerization; Electron Transport; Hydrogenase; Iron-Sulfur Proteins; Methanococcales; Models, Molecular; Molecular Sequence Data; NAD; Protein Structure, Quaternary; Protein Structure, Tertiary; Sequence Alignment; Structural Homology, Protein
PubMed: 16540118
DOI: 10.1016/j.jmb.2006.02.035 -
The Biochemical Journal Jan 19751. Lactate oxidase from Mycobacterium smegmatis is completely resolved into free flavin and apoenzyme by treatment with acid (NH4)2SO4. 2. Reconstitution involves rapid...
1. Lactate oxidase from Mycobacterium smegmatis is completely resolved into free flavin and apoenzyme by treatment with acid (NH4)2SO4. 2. Reconstitution involves rapid binding of FMN, but the recovery of enzyme activity was slower and appeared to be biphasic. 3. The preparation of the holoenzyme obtained differs from the native enzyme in specific activity, extinction coefficients and mobility on disc-gel electrophoresis. 4. Dialysis of this reconstituted enzyme in 0.1 M-sodium phosphate buffer, pH 7.0, at 0 degrees C for 1 week yields a preparation which closely resembles the native enzyme.
Topics: Alcohol Oxidoreductases; Ammonium Sulfate; Apoenzymes; Apoproteins; Electrophoresis, Polyacrylamide Gel; Flavin Mononucleotide; Kinetics; Lactates; Protein Binding; Spectrophotometry, Atomic
PubMed: 1191251
DOI: 10.1042/bj1450037 -
Biochemistry May 1998Membrane-integrated quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus was produced by heterologous expression of the gene for it in an Escherichia coli...
Membrane-integrated quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus was produced by heterologous expression of the gene for it in an Escherichia coli recombinant strain. The apoenzyme (lacking the cofactor pyrroloquinoline quinone, PQQ) was solubilized with Triton X-100 and purified to homogeneity. Reconstitution of the apoenzyme to full activity in the assay was achieved with a stoichiometric amount of PQQ in the presence of Mg2+. Just as for other PQQ-containing dehydrogenases where Ca2+ fulfills this role, Mg2+ anchors PQQ to the mGDH protein and activates the bound cofactor. This occurs in a precise way since high anomer specificity was found for the enzyme toward the sugars tested. Although the steady-state-type kinetics were as expected for a dye-linked dehydrogenase (ping-pong) and the PQQ in it was present in oxidized form, addition of glucose to the holoenzyme resulted in a very slow but continuous production of gluconolactone; i.e., the reaction did not stop after one turnover, with O2 apparently acting as an (albeit poor) electron acceptor by reoxidizing PQQH2 in the enzyme. The surprisingly low reactivity with glucose, in the absence of dye, as compared to the activity observed in the steady-state assay appeared to be due to formation of an anomalous enzyme form, mGDH. Formation of normal holoenzyme, mGDH, reducing added glucose immediately to gluconolactone (in one turnover), was achieved by treating mGDH with sulfite, by reconstituting apoenzyme with PQQ in the presence of sulfite, or by applying assay conditions to mGDH (addition of PMS/DCPIP). As compared to other quinoprotein dehydrogenases, mGDH appears to be unique with respect to the mode of PQQ-binding, as expressed by the special conditions for reconstitution and the absorption spectra of the bound cofactor, and the reactivity of the reduced enzyme toward O2. The primary cause for this seems not to be related to a different preference for the activating bivalent metal ion but to the special way of binding of PQQ to mGDH.
Topics: Animals; Apoenzymes; Bacterial Outer Membrane Proteins; Binding Sites; Cattle; Coenzymes; Glucose; Glucose Dehydrogenases; Kinetics; Oxidation-Reduction; PQQ Cofactor; Quinolones; Quinones; Recombinant Proteins
PubMed: 9578566
DOI: 10.1021/bi9722610