Purification, characterization, and mechanism of a flavin mononucleotide-dependent 2-nitropropane dioxygenase from Neurospora crassa

A nitroalkane-oxidizing enzyme was purified to homogeneity from Neurospora crassa. The enzyme is composed of two subunits; the molecular weight of each subunit is approximately 40,000. The enzyme catalyzes the oxidation of nitroalkanes to produce the corresponding carbonyl compounds. It acts on 2-nitropropane better than on nitroethane and 1-nitropropane, and anionic forms of nitroalkanes are much better substrates than are neutral forms. The enzyme does not act on aromatic compounds. When the enzyme reaction was conducted in an 18O2 atmosphere with the anionic form of 2-nitropropane as the substrate, acetone (with a molecular mass of 60 Da) was produced. This indicates that the oxygen atom of acetone was derived from molecular oxygen, not from water; hence, the enzyme is an oxygenase. The reaction stoichiometry was 2CH3CH(NO2)CH3 + O2-->2CH3COCH3 + 2HNO2, which is identical to that of the reaction of 2-nitropropane dioxygenase from Hansenula mrakii. The reaction of the Neurospora enzyme was inhibited by superoxide anion scavengers in the same manner as that of the Hansenula enzyme. Both of these enzymes are flavoenzymes; however, the Neurospora enzyme contains flavin mononucleotide as a prosthetic group, whereas the Hansenula enzyme contains flavin adenine dinucleotide.     

Purification and characterization of the catalytic subunit of protein phosphatase 1 from Neurospora crassa

Protein phosphorylation is a universal regulatory mechanism in eukaryotic cells. The phosphorylation state of proteins is affected by the antagonistic activities of protein kinases and phosphatases. Protein phosphatases (PPs) can be classified as serine/threonine and tyrosine specific phosphatases. Ser/Thr phosphatases are divided into four subclasses (PP1, PP2A, PP2B, PP2C) on the basis of their substrate specificity, metal ion dependence and inhibitor sensitivity. We were able to detect the activities of all four Ser/Thr protein phosphatases in the mycelial extract of Neurospora crassa. The catalytic subunit of PP1 was purified 1500-fold with a yield of 1.3% using ammonium sulfate-ethanol precipitation, DEAE-Sephacel, heparin-Sepharose and MonoQ FPLC chromatography. The protein product was nearly homogenous, as judged by SDS-polyacrylamide gel electrophoresis. The most important properties of the enzyme were the following: /1/ its molecular mass proved to be 35 kD, /2/ it was completely inhibited by inhibitor-2, microcystin and okadaic acid, /3/ it was bound to heparin-Sepharose, and /4/ its specific activity was 2000 mU/mg. These biochemical properties are very similar to those of the homologous enzyme from rabbit muscle and indicate a high level of conservation of PP1 structure during evolution.     

Purification and partial characterization of thioredoxin reductase from Streptomyces aureofaciens

Soluble carbonic anhydrase (CA, EC 4.2.1.1) inducible by low levels of CO2 was purified from the unicellular green alga Chlorella sorokiniana grown at alkaline pH. The purified CA had a specific activity of 2,300 units (mg protein)-1. The molecular mass of the CA was found to be 100 kDa by non-dissociating (native)-polyacrylamide gel electrophoresis and 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 50-kDa subunit was recognized by concanavalin A. These results suggest that the protein has a dimeric form with two 50-kDa subunits that are glycosylated in an asparagine-linked manner. The native CA was revealed by isoelectric focusing to be a very acidic protein with an isoelectric point of 4.2. About 60% of the CA activity was inhibited by 0.5 M NaCl. The enzyme was inactivated over 95% by preincubation with 50 mM dithiothreitol but not with 1 mM dithiothreitol. After partial amino acid sequence analysis, a cDNA clone of the CA was isolated and characterized. The cloned cDNA fragment encoded a 348-amino-acid polypeptide (36,709 Da) including an NH2-terminal hydrophobic signal peptide composed of 35 amino acids (3,725 Da). Conserved regions of sequences found in animal CAs, in the periplasmic (pCA) and the intracellular CAs of Chlamydomonas, and in the plasma-membrane-bound CA of Dunaliella (Dca) were also found in this Chlorella CA. The signal sequence was significantly homologous to the pCA and the Dca. The internal signal sequence between the large and the small subunits reported for pCA was not found in this Chlorella CA. The soluble CA of this alga was an alpha-type CA with salt-sensitive, periplasm-locating and acidic properties and very different from pCA and Dca with their salt-sensitive/neutral and salt-resistant/acidic properties, respectively.     

Isolation and characterization of a copper containing protein from blue cell walls of Neurospora crassa

Culturing Neurospora crassa in presence of toxic amounts of copper (0.63 mM) resulted in blue coloured mycelia and cell walls. Significant amounts (approximately 45%) of total mycelial copper were associated with cell wall isolates under conditions of copper toxicity. Hence, such blue cell walls were analysed to identify specific ligands involved in copper binding. While decuprification of the blue cell walls with 8-hydroxy quinoline (8 HQ) did not alter their copper binding abilities, similar treatment with EDTA (10 mM) decreased such abilities indicating that EDTA treatment lead to loss of copper binding ligands from cell walls. Treatment of blue cell walls with 8 HQ followed by EDTA resulted in the solubilization of a copper binding protein (relative MW approximately 14 kDa) which was associated with phosphate and carbohydrate moieties. On amino acid analysis, this protein was found to be devoid of free thiol groupings but enriched in acidic and basic amino acids, distinguishing it from classical intracellular metal binding proteins such as metallo-thioneins and phytochelatins that are inducively synthesized under conditions of metal toxicity. The biological significance of the isolated wall-bound copper binding protein, which appears to be a normal constituent of cell walls, is discussed in relation to cytoplasmic metal binding proteins and mechanism(s) adapted by fungi in countering metal toxicity.     

Purification and characterization of virginiamycin M1 reductase from Streptomyces virginiae

Virginiamycin M1 (VM1), produced by Streptomyces virginiae, is a polyunsaturated macrocyclic lactone antibiotic belonging to the virginiamycin A group. S. virginiae possesses an activity which stereospecifically reduces a 16-carbonyl group of VM1, resulting in antibiotically inactive 16R-dihydroVM1. The corresponding VM1 reductase was purified to homogeneity from crude extracts of S. virginiae in five steps, with 5,650-fold purification and 23% overall yield. The N-terminal amino acid sequence was determined to be MAIKLVIA. The purified enzyme showed an apparent Mr of 73,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an Mr of 280,000 by native molecular sieve high-performance liquid chromatography, indicating the tetrameric nature of the native enzyme. NADPH served as a coenzyme for the reduction, with a Km value of 0.13 mM, but NADH did not support the reaction, even at a concentration of 5 mM, indicating the NADPH-specific nature of the enzyme. The Km for VM1 was determined to be 1.5 mM in the presence of 2 mM NADPH. In the reverse reaction, only 16R-dihydroVM1, not the 16S-epimer, served as a substrate, with a less than 0.1% overall reaction rate compared to that of the forward reaction, confirming that the VM1 reductase participates solely in VM1 inactivation in vivo.     

Molecular modeling studies of the binding modes of aldose reductase inhibitors at the active site of human aldose reductase

Molecular modeling studies using the CHARMM method have been conducted to study the binding modes of aldose reductase inhibitors at the active site of aldose reductase. The energy minimized structures of aldose reductase with six structurally diverse inhibitors (spirofluorene-9,5'-imidazolidine-2',4'-dione (1), 9-fluoreneacetic acid (2), AL1576 (3), 2,7-difluoro-9-fluoreneacetic acid (4), FK366 (5), and Epalrestat (9)) indicate that the side chains of Tyr48, His110, and Trp111 can form numerous hydrogen bonds with either the carboxylate or the hydantoin group of the inhibitors while the side chains of Trp20, Trp111, and Phe122 are positioned to form aromatic-aromatic interactions. Of the three residues (Tyr 48, His 110, and Trp 111) that can form hydrogen bonds with the ionized portion of aldose reductase inhibitors, protonated His110 appears to play an important role in directing charged inhibitors to bind at the active site through charge interaction. Based on the binding mode of the inhibitors and their observed inhibitory activities, pharmacophore requirements for aldose reductase inhibitors are discussed.     

Coenzyme A disulfide reductase, the primary low molecular weight disulfide reductase from Staphylococcus aureus. Purification and characterization of the native enzyme

The human pathogen Staphylococcus aureus does not utilize the glutathione thiol/disulfide redox system employed by eukaryotes and many bacteria. Instead, this organism produces CoA as its major low molecular weight thiol. We report the identification and purification of the disulfide reductase component of this thiol/disulfide redox system. Coenzyme A disulfide reductase (CoADR) catalyzes the specific reduction of CoA disulfide by NADPH. CoADR has a pH optimum of 7.5-8.0 and is a dimer of identical subunits of Mr 49,000 each. The visible absorbance spectrum is indicative of a flavoprotein with a lambdamax = 452 nm. The liberated flavin from thermally denatured enzyme was identified as flavin adenine dinucleotide. Steady-state kinetic analysis revealed that CoADR catalyzes the reduction of CoA disulfide by NADPH at pH 7.8 with a Km for NADPH of 2 muM and for CoA disulfide of 11 muM. In addition to CoA disulfide CoADR reduces 4,4'-diphosphopantethine but has no measurable ability to reduce oxidized glutathione, cystine, pantethine, or H2O2. CoADR demonstrates a sequential kinetic mechanism and employs a single active site cysteine residue that forms a stable mixed disulfide with CoA during catalysis. These data suggest that S. aureus employs a thiol/disulfide redox system based on CoA/CoA-disulfide and CoADR, an unorthodox new member of the pyridine nucleotide-disulfide reductase superfamily.     

Purification and characterization of recombinant Thermotoga maritima dihydrofolate reductase

We have overexpressed the gene for dihydrofolate reductase (DHFR) from Thermotoga maritima in Escherichia coli and characterized the biochemical properties of the recombinant protein. This enzyme is involved in the de novo synthesis of deoxythymidine 5'-phosphate and is critical for cell growth. High levels of T. maritima DHFR in the new expression system conferred resistance to high levels of DHFR inhibitors which inhibit the growth of non-recombinant cells. The enzyme was purified to homogeneity in the following two steps: heat treatment followed by affinity chromatography or cation-exchange chromatography. Most of the biochemical properties of T. maritima DHFR resemble those of other bacterial or eukaryotic DHFRs, however, some are unique to T. maritima DHFR. The pH optima for activity, Km for substrates, and polypeptide chain length of T. maritima DHFR are similar to those of other DHFRs. In addition, the secondary structure of T. maritima DHFR, as measured by circular dichroism, is similar to that of other DHFRs. Interestingly, T. maritima DHFR exhibits some characteristics of eukaryotic DHFRs, such as a basic pI, an excess of positively charged residues in the polypeptide chain and activation of the enzyme by inorganic salts and urea. Unlike most other DHFRs which are monomeric or part of a bifunctional DHFR-thymidylate synthase (TS) enzyme, T. maritima DHFR seems to generally form a dimer in solution and is also much more thermostable than other DHFRs. It may be that dimer formation is a key factor in determining the stability of T. maritima DHFR.     

Indications for the occurrence of nitric oxide synthases in fungi and plants and the involvement in photoconidiation of Neurospora crassa

The structure of the complex of Aeromonas proteolytica aminopeptidase, a two-zinc exopeptidase, with the inhibitor p-iodo-D-phenylalanine hydroxamate has been determined by X-ray crystallography. Refinement of the structure, which includes 220 water molecules, using data at 0.80-0.23-nm resolution resulted in a crystallographic residual R value of 16%. The hydroxamate group adopts a planar conformation whereby the two oxygen atoms interact with the zinc ions. The N-hydroxyl group of the inhibitor is located between the two zinc ions, a position which is close to that occupied by a water molecule in the native structure. The carbonyl oxygen of the inhibitor binds to Zn1, which becomes pentacoordinated while Zn2 remains tetracoordinated, in contrast to the native protein where both zinc ions were shown to be tetracoordinated and structurally equivalent. Interactions of the carboxylate oxygens of Glu151 with the hydroxamate group play an important role in the stabilization of the complex.     

Purification and characterization of the respiratory arsenate reductase of Chrysiogenes arsenatis

GS 4071 is a potent carbocyclic transition-state analog inhibitor of influenza virus neuraminidase with activity against both influenza A and B viruses in vitro. GS 4116, the guanidino analog of GS 4071, is a 10-fold more potent inhibitor of influenza virus replication in tissue culture than GS 4071. In this study we determined the oral bioavailabilities of GS 4071, GS 4116, and their respective ethyl ester prodrugs in rats. Both parent compounds and the prodrug of the guanidino analog exhibited poor oral bioavailability (2 to 4%) and low peak concentrations in plasma (Cmaxs; Cmax <0.06 microg/ml). In contrast, GS 4104, the ethyl ester prodrug of GS 4071, exhibited good oral bioavailability (35%) as GS 4071 and high Cmaxs of GS 4071 (Cmax = 0.47 microg/ml) which are 150 times the concentration necessary to inhibit influenza virus neuraminidase activity by 90%. The bioavailability of GS 4104 as GS 4071 was also determined in mice (30%), ferrets (11%), and dogs (73%). The plasma of all four species exhibited high, sustained concentrations of GS 4071 such that at 12 h postdosing the concentrations of GS 4071 in plasma exceeded those necessary to inhibit influenza virus neuraminidase activity by 90%. These results demonstrate that GS 4104 is an orally bioavailable prodrug of GS 4071 in animals and that it has the potential to be an oral agent for the prevention and treatment of influenza A and B virus infections in humans.     

Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase

Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3- null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.     

Structural studies of binding site tryptophan mutants in the high-affinity streptavidin-biotin complex

Previous thermodynamic and computational studies have pointed to the important energetic role of aromatic contacts in generating the exceptional binding free energy of streptavidin-biotin association. We report here the crystallographic characterization of single site tryptophan mutants in investigating structural consequences of alterations in these aromatic contacts. Four tryptophan residues, Trp79, Trp92, Trp108 and Trp120, play an important role in the hydrophobic binding contributions, which along with a hydrogen bonding network and a flexible binding loop give rise to tight ligand binding (Ka approximately 10(13) M-1). The crystal structures of ligand-free and biotin-bound mutants, W79F, W108F, W120F and W120A, in the resolution range from 1.9 to 2.3 A were determined. Nine data sets for these four different mutants were collected, and structural models were refined to R-values ranging from 0.15 to 0.20. The major question addressed here is how these mutations influence the streptavidin binding site and in particular how they affect the binding mode of biotin in the complex. The overall folding of streptavidin was not significantly altered in any of the tryptophan mutants. With one exception, only minor deviations in the unbound structures were observed. In one crystal form of unbound W79F, there is a coupled shift in the side-chains of Phe29 and Tyr43 toward the mutation site, although in a different crystal form these shifts are not observed. In the bound structures, the orientation of biotin in the binding pocket was not significantly altered in the mutant complex. Compared with the wild-type streptavidin-biotin complex, there were no additional crystallographic water molecules observed for any of the mutants in the binding pocket. These structural studies thus suggest that the thermodynamic alterations can be attributed to the local alterations in binding residue composition, rather than a rearrangement of binding site architectures.     

Purification and characterization of a human bradykinin binding protein from inflammatory cells

Bradykinin (BK) is a potent mediator with a broad spectrum of pharmacological and inflammatory actions which are exerted through cell surface receptors. We report here the affinity chromatographic purification of a novel 14 kDa BK binding protein from human blood neutrophils and also peripheral blood mononuclear cells (PBMC), 80% of which are lymphocytes. Radioreceptor crosslinking experiments using bifunctional crosslinkers and radiolabelled BK identified a 14 kDa protein in these cell types both on the cell surface, in glycerol purified plasma membranes and in detergent solubilized cell extracts. Purification by BK affinity chromatography from a variety of BK responsive human cell types i.e. CCD-16Lu lung fibroblasts, HL60 promyelocytes, U937 myelomonocytes and Jurkat T lymphocytes also demonstrated a 14 kDa protein. Purified material obtained from three different BK affinity columns all demonstrated three major proteins at 190, 50 and 14 kDa when eluted with either excess BK or mild acid. Neutrophil fractions from detergent solubilized cell extracts contained an additional 150 kDa protein when eluted with mild acid. Neutrophil and PBMC crude plasma membrane BK affinity column purifications yielded only a single 14 kDa protein. Radioreceptor dot assays of the purified neutrophil eluates containing the 14 kDa protein revealed specific binding to [125I]-BK with a 160 fold excess signal ratio over the original membrane extract. Our data indicates that we have successfully isolated a 14 kDa novel human BK specific binding protein expressed on the surface of inflammatory cells.     

Spectroscopic, kinetic, and electrochemical characterization of heterologously expressed wild-type and mutant forms of copper-containing nitrite reductase from Rhodobacter sphaeroides 2.4.3

We report the development of a high-yield heterologous expression system for the copper-containing nitrite reductase from a denitrifying variant of Rhodobacter sphaeroides. Typical yields of wild-type protein are 20 mg L-1, which can be fully loaded with copper. Nitrite reductase contains an unusual blue-green Type 1 copper center with a redox/electron transfer function and a nearby Type 2 center where nitrite binds and is reduced to nitric oxide. The wild-type enzyme was characterized by: (1) its blue-green Type 1 optical spectrum; (2) its EPR spectrum showing rhombic character to its Type 1 center and nitrite perturbation to its Type 2 center; (3) its 247-mV Type 1 midpoint potential which is low relative to other Type 1 centers; and (4) its kinetics as measured by both steady-state and stopped-flow methods. The Type 2 copper reduction potential as monitored by EPR in the absence of nitrite was below 200 mV so that reduction of the Type 2 center by the Type 1 center in the absence of nitrite is not energetically favored. The mutation M182T in which the methionine ligand of Type 1 copper was changed to a threonine resulted in a blue rather than blue-green Type 1 center, a midpoint potential that increased by more than 100 mV above that of the wild-type Type 1 center, and a somewhat reduced nitrite reductase activity. The blue color and midpoint potential of M182T are reminiscent of plastocyanin, but the Type 1 cupric HOMO ground-state electronic g value and copper hyperfine properties of M182T (as well as cysteine and histidine ENDOR hyperfine properties; see next paper) were unchanged from those of the blue-green native Type 1 center. His287 is a residue in the Type 2 region whose imidazole ring was thought to hydrogen bond to the Type 2 axial ligand but not directly to Type 2 copper. The mutation H287E resulted in a 100-fold loss of enzyme activity and a Type 2 EPR spectrum (as well as ENDOR spectra; see next paper) which were no longer sensitive to the presence of nitrite.     

M-glycogenin, the protein moiety of Neurospora crassa proteoglycogen, is an auto- and transglucosylating enzyme

Neurospora crassa proteoglycogen was purified and its protein moiety, M-glycogenin, was released by amylolytic treatment. The released protein was capable of autoglucosylation from UDP-glucose forming glucosyl-alpha 1,4-glucosyl linkage. The kinetics of autoglucosylation suggested an intramolecular mechanism of reaction. M-glycogenin was also able to glucosylate dodecyl-beta-maltoside and autoglucosylate, simultaneously and independently. Both auto- and transglucosylation reactions were dependent on Mn2+. Thus, M-glycogenin, which has also been described as the constituent of Escherichia coli proteoglycogen (A. Goldraij and J. A. Curtino. 1993, Biochem. Mol. Biol. Int. 30, 453-458), is a glucosyltransferase that bears similar catalytic properties with mammalian glycogenin. This is the first report on the enzymatic character of the protein constituent of proteoglycogen in primitive organisms, which suggest that the mechanism for the de novo biosynthesis of glycogen was conserved over a very long period of evolution.     

Purification, characterization and molecular cloning of TGP1, a novel G-DNA binding protein from Tetrahymena thermophila

G-DNA, a polymorphic family of four-stranded DNA structures, has been proposed to play roles in a variety of biological processes including telomere function, meiotic recombination and gene regulation. Here we report the purification and cloning of TGP1, a G-DNA specific binding protein from Tetrahymena thermophila. TGP1 was purified by three-column chromatographies, including a G-DNA affinity column. Two major proteins (approximately 80 and approximately 40 kDa) were present in the most highly purified column fraction. Renaturation experiments showed that the approximately 80 kDa protein contains TGP1 activity. Biochemical characterization showed that TGP1 is a G-DNA specific binding protein with a preference for parallel G-DNAs. The TGP1/DNA complex has a dissociation constant (Kd) of approximately 2.2 x 10(-8) M and TGP1 can form supershift in gel mobility shift assays. The cDNA coding TGP1 was cloned and sequenced based upon an internal peptide sequence obtained from the approximately 80 kDa protein. Sequence analyses showed that TGP1 is a basic protein with a pI of 10.58, and contains two extensively hydrophilic and basic domains. Homology searches revealed that TGP1 is a novel protein sharing weak similarities with a number of proteins.     

Isolation and characterization of a new gene, sre, which encodes a GATA-type regulatory protein that controls iron transport in Neurospora crassa

Multiple GATA factors - regulatory proteins with consensus zinc finger motifs that bind to DNA elements containing a GATA core sequence - exist in the filamentous fungus Neurospora crassa. One GATA factor, NIT2. controls nitrogen metabolism, whereas two others, WC-1 and WC-2, regulate genes responsive to blue light induction. A gene encoding a new GATA factor, named SRE, was isolated from Neurospora using a PCR-mediated method. Sequence analysis of the new GATA factor gene revealed an ORF specifying 587 amino acids, which is interrupted by two small introns. Unlike all previously known Neurospora GATA factors, which possess a single zinc-finger DNA-binding motif, SRE contains two GATA-type zinc fingers. The deduced amino acid sequence of SRE shows significant similarity to URBSI of Ustilago and SREP of Penicillium. A loss-of-function mutation was created by the RIP procedure. Analysis of sre+ and sre- strains revealed that SRE acts as a negative regulator of iron uptake in Neurospora by controlling the synthesis of siderophores. Siderophore biosynthesis is repressed by high iron concentrations in the wild-type strain but not in sre- mutant cells. The sre promoter contains a number of GATA sequences; however, expression of sre mRNA occurs in a constitutive fashion and is not regulated by the concentration of iron available to the cells.