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1.
While most protist mitochondrial enzymes could be identified in database, the membrane anchor subunits of Complex II and FoF1-ATP synthase of malaria parasites are not annotated. Based on the presence of structural fingerprints or proteomics data from other protists, here we present their candidates. In contrast to canonical subunits, Plasmodium Complex II anchors have two transmembrane helices and may coordinate heme b via Tyr in place of His. Transmembrane helix IV of ATP synthase subunit a lacks an essential Arg residue. Membrane anchors of Plasmodium Complex II and ATP synthase are divergent from orthologs and promising targets for new chemotherapeutics. 相似文献
2.
The study of the thermodynamic redox behavior of the hemes from two members of the A family of heme-copper oxygen reductases, Paracoccus denitrificans aa3 (A1 subfamily) and Rhodothermus marinus caa3 (A2 subfamily) enzymes, is presented. At different pH values, midpoint reduction potentials and interaction potentials were obtained in the framework of a pairwise model for two interacting redox centers. In both enzymes, the hemes have different reduction potentials. For the A1-type enzyme, it was shown that heme a has a pH-dependent midpoint reduction potential, whereas that of heme a3 is pH independent. For the A2-type enzyme the opposite was observed. The midpoint reduction potential of heme c from subunit II of the caa3 enzyme was determined by fitting the data with a single-electron Nernst curve, and it was shown to be pH dependent. The results presented here for these A-type enzymes are compared with those previously obtained for representative members of the B and C families. 相似文献
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《Phytochemistry》1987,26(3):655-657
Five phosphatases were isolated from the latices of three members of the Euphorbiaceae. From Euphorbia lathyris were obtained phosphatases 11 and 12; from E. trigona phosphatase t and from Elaeophorbia drupifera the enzymes d1 and d2. Phosphatases 11, 12 and t were purified to homogeneity. Amino acid compositions are reported and other properties of the enzymes are described. The two enzymes described from E. lathyris both have two pH maxima d(11 at 5.0 and 6.8,12 at 5.8 and 7.5) while t, d1 and d2 respectively have maxima at pHs of 5.6,5.6 and 5.0. On the basis of their responses to several residue-specific inhibitors the five phosphatases apparently comprise three groups: 12 and d1, t and d2, and 11. 相似文献
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Discovery of natural nicking endonucleases Nb.BsrDI and Nb.BtsI and engineering of top-strand nicking variants from BsrDI and BtsI 总被引:1,自引:1,他引:0
Xu SY Zhu Z Zhang P Chan SH Samuelson JC Xiao J Ingalls D Wilson GG 《Nucleic acids research》2007,35(14):4608-4618
BsrDI and BtsI restriction endonucleases recognize and cleave double-strand DNA at the sequences GCAATG (2/0) and GCAGTG (2/0), respectively. We have purified and partially characterized these two enzymes, and analyzed the genes that encode them. BsrDI and BtsI are unusual in two respects: each cleaves DNA as a heterodimer of one large subunit (B subunit) and one small subunit (A subunit); and, in the absence of their small subunits, the large subunits behave as sequence-specific DNA nicking enzymes and only nick the bottom strand of the sequences at these respective positions: GCAATG (−/0) and GCAGTG (−/0). We refer to the single subunit, the bottom-strand nicking forms as ‘hemidimers’. Amino acid sequence comparisons reveal that BsrDI and BtsI belong to a family of restriction enzymes that possess two catalytic sites: a canonical PD-Xn-EXK and a second non-canonical PD-Xn-E-X12-QR. Interestingly, the other family members, which include BsrI (ACTGG 1/−1) and BsmI/Mva1269I (GAATGC 1/−1) are single polypeptide chains, i.e. monomers, rather than heterodimers. In BsrDI and BtsI, the two catalytic sites are found in two separate subunits. Site-directed mutagenesis confirmed that the canonical catalytic site located at the N-terminus of the large subunit is responsible for the bottom-strand cleavage, whereas the non-canonical catalytic site located in the small subunit is responsible for hydrolysis of the top strand. Top-strand specific nicking variants, Nt.BsrDI and Nt.BtsI, were successfully engineered by combining the catalytic-deficient B subunit with wild-type A subunit. 相似文献
7.
There are two 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) in rat liver, one in mitochondria (type I enzyme), and another in peroxisomes (type II enzyme). In a series of the studies on the properties and the physiological roles of fatty acid oxidation systems in both organelles, the two enzymes were purified and compared for their properties. The final preparations obtained were judged to be homogeneous based on the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sedimentation velocity analysis. Type I enzyme was composed of two identical subunits of molecular weight of 32,000, whereas type II enzyme was a monomeric enzyme having a molecular weight of 70,000–77,000. These subunit structures were confirmed by the results of fluorescence studies. Both enzymes were different in amino acid compositions, especially in the contents of tryptophan and half-cystine. Antibodies against them formed single precipitin lines for the corresponding enzymes, but not for the others when subjected to an Ouchterlony double-diffusion test. The Km values of type II enzyme for various substrates were lower than those of type I enzyme except those for acetoacetyl-CoA. As for 3-hydroxyacyl-CoA substrates, both enzymes had lower Km's for longer-chain substrates. The V for the substrates of C4C10 were similar for each enzyme, though the value of type II enzyme for C10 substrate was rather lower. The results of fluorescence studies suggested that their dissociation constants for NADH were lower and those for NAD+ were higher at lower pH. Both enzymes were specific to l-form of 3-hydroxyacyl-CoA substrate. The optimal pH of the forward reaction of type I and type II enzymes was 9.6 and 9.8, and of the reverse reaction, 4.5 and 6.2, respectively. From these results they were concluded to be completely different enzymes. 相似文献
8.
Purification and Molecular and Immunological Characterization of a Unique Phosphoribulokinase from the Green Alga Selenastrum minutum
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A unique phosphoribulokinase (ADP:D-ribulose 5-phosphate 1-phosphotransferase, EC 2.7.1.19) has been purified to homogeneity from the green alga Selenastrum minutum. The enzyme has a native molecular mass of about 83 kilodaltons and a native isoelectric point of 5.1. The enzyme consists of two different-sized subunits of 41 and 40 kilodaltons, implying that it is a heterodimer. This is the first report of a eukaryotic heterodimeric phosphoribulokinase. The in vivo existence of two nonidentical subunits of S. minutum phosphoribulokinase was confirmed by western blot analysis of crude protein extracts from trichloroacetic acid-killed cells. These two subunits were immunologically similar, as rabbit immunoglobulin G affinity purified against the 41 kilodalton subunit of S. minutum phosphoribulokinase (PRK) cross-reacts with the 40 kilodalton subunit and vice versa. Antibodies against S. minutum phosphoribulokinase also cross-react with the spinach enzyme. NH2-terminal sequencing revealed that the two S. minutum PRK subunits shared a considerable degree of structure homology with each other and with the enzymes from spinach and Chlamydomonas reinhardtii, but not with PRK from Rhodobacter sphaeroides. There are, however, differences between the NH2-terminal amino acid sequences of the two S. minutum PRK subunits, that imply that they are the products of separate genes or products of two different mRNAs spliced from a single gene. 相似文献
9.
Sung-Koo Kang Jong-Ho Lee Young-Choon Lee Cheorl-Ho Kim 《Biochimica et Biophysica Acta (BBA)/General Subjects》2006
Isonicotinic acid hydrazide (Isoniazid, INH) is one of the major drugs worldwide used in the chemotherapy of tuberculosis. Many investigators have emphasized that INH activation is associated with mycobacterial catalase-peroxidase (katG). However, INH activation mechanism is not completely understood. In this study, katG of M. bovis BCG was separated and purified into two katGs, katG I (named as relatively higher molecular weight than katG II) and katG II, indicating that there is some difference in protein structure between two katGs. The molecular weight of the enzymes of katG I and katG II was estimated to be approximately 150,000 Da by gel filtration, and its subunit was 75,000 Da as determined by SDS-PAGE, indicating that purified enzyme was composed of two identical subunits. The specific activity of the purified enzyme katG I was 991.1 (units/mg). The enzymes were then investigated in INH activation by using gas chromatography mass spectrometry (GC-MS). The analysis of GC-MS showed that the katG I from M. bovis BCG directly converted INH (Mr, 137) to isonicotinamide (Mr, 122), not to isonicotinic acid (Mr, 123), in the presence or absence of H2O2. Therefore, this is the first report that katG I, one of two katGs with almost same molecular weight existed in M. bovis BCG, converts INH to isonicotinamide and this study may give us important new light on the activation mechanism of INH by KatG between M. bovis BCG and M. tuberculosis. 相似文献
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《BBA》2020,1861(7):148188
Ni-containing CO-dehydrogenases (CODHs) allow some microorganisms to couple ATP synthesis to CO oxidation, or to use either CO or CO2 as a source of carbon. The recent detailed characterizations of some of them have evidenced a great diversity in terms of catalytic properties and resistance to O2. In an effort to increase the number of available CODHs, we have heterologously produced in Desulfovibrio fructosovorans, purified and characterized the two CooS-type CODHs (CooS1 and CooS2) from the hyperthermophilic archaeon Thermococcus sp. AM4 (Tc). We have also crystallized CooS2, which is coupled in vivo to a hydrogenase. CooS1 and CooS2 are homodimers, and harbour five metalloclusters: two [Ni4Fe-4S] C clusters, two [4Fe-4S] B clusters and one interfacial [4Fe-4S] D cluster. We show that both are dependent on a maturase, CooC1 or CooC2, which is interchangeable. The homologous protein CooC3 does not allow Ni insertion in either CooS. The two CODHs from Tc have similar properties: they can both oxidize and produce CO. The Michaelis constants (Km) are in the microM range for CO and in the mM range (CODH 1) or above (CODH 2) for CO2. Product inhibition is observed only for CO2 reduction, consistent with CO2 binding being much weaker than CO binding. The two enzymes are rather O2 sensitive (similarly to CODH II from Carboxydothermus hydrogenoformans), and react more slowly with O2 than any other CODH for which these data are available. 相似文献
12.
Iizuka R Sugano Y Ide N Ohtaki A Yoshida T Fujiwara S Imanaka T Yohda M 《Journal of molecular biology》2008,377(3):972-983
Prefoldin is a heterohexameric molecular chaperone complex that is found in the eukaryotic cytosol and also in archaea. It captures a nonnative protein and subsequently delivers it to a group II chaperonin for proper folding. Archaeal prefoldin is a heterocomplex containing two α subunits and four β subunits with the structure of a double β-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles. We have studied the protein folding mechanism of group II chaperonin using those of Thermococcus sp. strain KS-1 (T. KS-1) because they exhibit high protein folding activity in vitro. We have also demonstrated functional cooperation between T. KS-1 chaperonins and prefoldin from Pyrococcus horikoshii OT3. Recent genome analysis has shown that Thermococcus kodakaraensis KOD1 contains two pairs of prefoldin subunit genes, correlating with the existence of two different chaperonin subunits. In this study, we characterized four different recombinant prefoldin complexes composed of two pairs of prefoldin subunits (α1, α2, β1, and β2) from T. KS-1. All of them (α1-β1, α2-β1, α1-β2, and α2-β2) exist as α2β4 heterohexamers and can protect several proteins from forming aggregates with different activities. We have also compared the collaborative activity between the prefoldin complexes and the cognate chaperonins. Prefoldin complexes containing the β1 subunit interacted with the chaperonins more strongly than those with the β2 subunit. The results suggest that Thermococcus spp. express different prefoldins for different substrates or conditions as chaperonins. 相似文献
13.
The active site tyrosine residue of all monomeric type IB topoisomerases resides in the C-terminal domain of the enzyme. Leishmania donovani, possesses unusual heterodimeric type IB topoisomerase. The small subunit harbors the catalytic tyrosine within the SKXXY motif. To explore the functional relationship between the two subunits, we have replaced the small subunit of L.donovani topoisomerase I with a C-terminal fragment of human topoisomerase I (HTOP14). The purified LdTOP1L (large subunit of L.donovani topoisomerase I) and HTOP14 were able to reconstitute topoisomerase I activity when mixed in vitro. This unusual enzyme, ‘LeishMan’ topoisomerase I (Leish for Leishmania and Man for human) exhibits less efficiency in DNA binding and strand passage compared with LdTOP1L/S. Fusion of LdTOP1L with HTOP14 yielded a more efficient enzyme with greater affinity for DNA and faster strand passage ability. Both the chimeric enzymes are less sensitive to camptothecin than LdTOP1L/S. Restoration of topoisomerase I activity by LdTOP1L and HTOP14 suggests that the small subunit of L.donovani topoisomerase I is primarily required for supplying the catalytic tyrosine. Moreover, changes in the enzyme properties due to substitution of LdTOP1S with HTOP14 indicate that the small subunit contributes to subunit interaction and catalytic efficiency of the enzyme. 相似文献
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Jinyou Zhang Xupeng Cao Yanjuan Xin Song Xue Wei Zhang 《World journal of microbiology & biotechnology》2013,29(10):1791-1799
2-haloacid dehalogenases are enzymes that are capable of degrading 2-haloacid compounds. These enzymes are produced by bacteria, but so far they have only been purified and characterized from terrestrial bacteria. The present study describes the purification and characterization of 2-haloacid dehalogenase from the marine bacterium Pseudomonas stutzeri DEH130. P. Stutzeri DEH130 contained two kinds of 2-haloacid dehalogenase (designated as Dehalogenase I and Dehalogenase II) as detected in the crude cell extract after ammonium sulfate fractionation. Both enzymes appeared to exhibit stereo-specificity with respect to substrate. Dehalogenase I was a 109.9-kDa enzyme that preferentially utilized D-2-chloropropropionate and had optimum activity at pH 7.5. Dehalogenase II, which preferentially utilized L-2-chloropropionate, was further purified by ion-exchange chromatography and gel filtration. Purified Dehalogenase II appeared to be a dimeric enzyme with a subunit of 26.0-kDa. It had maximum activity at pH 10.0 and a temperature of 40 °C. Its activity was not inhibited by DTT and EDTA, but strongly inhibited by Cu2+, Zn2+, and Co2+. The K m and V max for L-2-chloropropionate were 0.3 mM and 23.8 μmol/min/mg, respectively. Its substrate specificity was limited to short chain mono-substituted 2-halocarboxylic acids, with no activity detected toward fluoropropionate and monoiodoacetate. This is the first report on the purification and characterization of 2-haloacid dehalogenase from a marine bacterium. 相似文献
16.
Involvement of the Terminal Oxygenase β Subunit in the Biphenyl Dioxygenase Reactivity Pattern toward Chlorobiphenyls
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Biphenyl dioxygenase (BPH dox) oxidizes biphenyl on adjacent carbons to generate 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356 and in Pseudomonas sp. strain LB400. The enzyme comprises a two-subunit (α and β) iron sulfur protein (ISPBPH), a ferredoxin (FERBPH), and a ferredoxin reductase (REDBPH). B-356 BPH dox preferentially catalyzes the oxidation of the double-meta-substituted congener 3,3′-dichlorobiphenyl over the double-para-substituted congener 4,4′-dichlorobiphenyl or the double-ortho-substituted congener 2,2′-dichlorobiphenyl. LB400 BPH dox shows a preference for 2,2′-dichlorobiphenyl, and in addition, unlike B-356 BPH dox, it can catalyze the oxidation of selected chlorobiphenyls such as 2,2′,5,5′-tetrachlorobiphenyl on adjacent meta-para carbons. In this work, we examine the reactivity pattern of BPH dox toward various chlorobiphenyls and its capacity to catalyze the meta-para dioxygenation of chimeric enzymes obtained by exchanging the ISPBPH α or β subunit of strain B-356 for the corresponding subunit of strain LB400. These hybrid enzymes were purified by an affinity chromatography system as His-tagged proteins. Both types, the chimera with the α subunit of ISPBPH of strain LB400 and the β subunit of ISPBPH of strain B-356 (the αLB400βB-356 chimera) and the αB-356βLB400 chimera, were functional. Results with purified enzyme preparations showed for the first time that the ISPBPH β subunit influences BPH dox’s reactivity pattern toward chlorobiphenyls. Thus, if the α subunit were the sole determinant of the enzyme reactivity pattern, the αB-356βLB400 chimera should have behaved like B-356 ISPBPH; instead, its reactivity pattern toward the substrates tested was similar to that of LB400 ISPBPH. On the other hand, the αLB400βB-356 chimera showed features of both B-356 and LB400 ISPBPH where the enzyme was able to metabolize 2,2′- and 3,3′-dichlorobiphenyl and where it was able to catalyze the meta-para oxygenation of 2,2′,5,5′-tetrachlorobiphenyl. 相似文献
17.
P. H. Benetti S. I. Kim M. Canonge T. Chardot J. C. Meunier 《Molecular genetics and genomics : MGG》1997,256(4):355-364
Casein kinase II from the yeast Yarrowia lipolytica is a heterotetramer of the form αα′β2. We report on the cloning and sequencing of a partial cDNA and of the complete genomic DNA coding for the catalytic α subunit of the casein kinase II from this yeast species. The sequence of the gene coding for this enzyme has been analyzed. No intron was found in the gene, which is present in a single copy. The deduced amino acid sequence of the gene shows high similarity with those of α subunit described in other species, although, uniquely, Y. lipolytica CKIIα lacks cysteines. We find that the α subunit sequence of Y. lipolytica CKII is shown greater homology with the corresponding protein from S. pombe than with that from S. cerevisiae. We have analyzed CKIIα expression and CKIIα activity. We show that expression of this enzyme is regulated. The catalytic subunit is translated from a single mRNA, and the enzyme is present at a very low level in Y. lipolytica, as in other yeasts. 相似文献
18.
Stoll L Hall J Van Buren N Hall A Knight L Morgan A Zuger S Van Deusen H Gentile L 《Biophysical journal》2007,92(4):1343-1349
Ionotropic glutamate receptors (iGluRs), a family of ligand-gated ion channels, are responsible for the majority of fast excitatory neurotransmission in the central nervous system. Within this family, different members serve distinct roles at glutamatergic synapses. Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors mediate fast depolarization while N-methyl-D-aspartate (NMDA) receptors mediate the slower component of the excitatory postsynaptic potential. These disparate functions suggest alternate modes of regulation. In this work, we show that endogenous regulators of iGluRs have different abilities to bind to specific domains of NMDA NR1-1b and AMPA GluR2 subunits. We have previously shown that the sulfated neurosteroids pregnenolone sulfate and 3α-hydroxy-5β-pregnan-20-one sulfate bind to the extracellular glutamate-binding core (S1S2) of the GluR2 subunit. Here we show that neither neurosteroid binds to the S1S2 domain of the NMDA NR1-1b subunit. This NR1-1b NMDA domain does, however, bind to the endogenous polyamines spermine and spermidine as well as Zn(II). Binding of the polyamines and Zn(II) to the S1S2 domain of the GluR2 subunit was not observed. This binding of Zn(II) and polyamines to the S1S2 domain of the NR1-1b subunit defines a new binding site for each of these modulators. 相似文献
19.
《Inorganica chimica acta》1988,141(1):145-149
This contribution reports the synthesis and characterization of the organothorium alkylthiolate complex [(CH3)5C5]2Th(SCH2CH2CH3)2. This compound crystallizes in the monoclinic space group C2/c (#15) with four molecules in a cell of dimensions a=19.066(2), b=11.603(1), c=16.379(2) Å, and β=130.08(1)°. Least-squares refinement led to a value for the conventional R index (on Fo) of 0.040 for 132 variables and 2030 observations having Fo2⩾3σ(Fo2). The molecular structure consists of an unexceptional ‘bent sandwich’ [(CH3)5C5]2Th fragment coordinated to two n-propylthiolate ligands. The ThS bond distance is 2.718(3) Å; the SC(α) distance, 1.78(2) Å; the ThSC(α) angle, 108.3(5)°; and the SThS′ angle, 102.5(2)°. Contrasts are drawn with the structures of analogous actinide alkoxides 相似文献
20.
Grigorios A. Papadakos Cynthia M. Dupureur 《Journal of biological inorganic chemistry》2011,16(8):1269-1278
Understanding the roles of metal ions in restriction enzymes has been complicated by both the presence of two metal ions in
many active sites and their homodimeric structure. Using a single-chain form of the wild-type restriction enzyme PvuII (scWT) in which subunits are fused with a short polypeptide linker (Simoncsits et al. in J. Mol. Biol. 309:89–97, 2001), we have characterized metal ion and DNA binding behavior in one subunit and examined the effects of the linker on dimer
behavior. scWT exhibits heteronuclear single quantum coherence NMR spectra similar to those of native wild-type PvuII (WT). For scWT, isothermal titration calorimetry data fit to two Ca(II) sites per subunit with low-millimolar K
ds. The variant scWT|E68A, in which metal ion binding in one subunit is abolished by mutation, also binds two Ca(II) ions in
the WT subunit with low-millimolar K
ds. When there are no added metal ions, DNA binding affinity for scWT is tenfold stronger than that of the native WT, but tenfold
weaker at saturating Ca(II) concentration. In the presence of Ca(II), scWT|E68A binds target DNA similarly to scWT, indicating
that high-affinity substrate binding can be carried energetically by one metal-ion-binding subunit. Global analysis of DNA
binding data for scWT|E68A suggests that the metal-ion-dependent behaviors observed for WT are reflective of independent subunit
behavior. This characterization provides an understanding of subunit contributions in a homodimeric context. 相似文献