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Biomolecules Aug 2018RNA modifications have been implicated in diverse and important roles in all kingdoms of life with over 100 of them present on tRNAs. A prominent modification at the...
RNA modifications have been implicated in diverse and important roles in all kingdoms of life with over 100 of them present on tRNAs. A prominent modification at the wobble base of four tRNAs is the 7-deaza-guanine derivative queuine which substitutes the guanine at position 34. This exchange is catalyzed by members of the enzyme class of tRNA guanine transglycosylases (TGTs). These enzymes incorporate guanine substituents into tRNA, tRNA tRNA, and tRNA in all kingdoms of life. In contrast to the homodimeric bacterial TGT, the active eukaryotic TGT is a heterodimer in solution, comprised of a catalytic QTRT1 subunit and a noncatalytic QTRT2 subunit. Bacterial TGT enzymes, that incorporate a queuine precursor, have been identified or proposed as virulence factors for infections by pathogens in humans and therefore are valuable targets for drug design. To date no structure of a eukaryotic catalytic subunit is reported, and differences to its bacterial counterpart have to be deducted from sequence analysis and models. Here we report the first crystal structure of a eukaryotic QTRT1 subunit and compare it to known structures of the bacterial TGT and murine QTRT2. Furthermore, we were able to determine the crystal structure of QTRT1 in complex with the queuine substrate.
Topics: Apoenzymes; Catalytic Domain; Crystallography, X-Ray; Guanine; Humans; Models, Molecular; Pentosyltransferases
PubMed: 30149595
DOI: 10.3390/biom8030081 -
The International Journal of... 19901. An apo-NADPH-adreno-ferredoxin reductase (EC 1.18.1.2) was obtained from bovine adrenocortical mitochondria and its physicochemical properties were investigated. 2....
1. An apo-NADPH-adreno-ferredoxin reductase (EC 1.18.1.2) was obtained from bovine adrenocortical mitochondria and its physicochemical properties were investigated. 2. The effects of various substances such as NADPH, FAD and adreno-ferredoxin on the interaction of the apo-reductase were investigated by various column chromatographies. 3. The apo- and holo-reductases were found to be separated by adreno-ferredoxin affinity chromatography. 4. The removal of FAD from NADPH-adreno-ferredoxin reductase did not affect the net charge of the reductase. 5. The values of s20,w of apo- and holo-reductases were 3.8 x 10(-13) sec and 3.9 x 10(-13) sec, respectively. 6. The apo-reductase was more easily denatured by heat treatment than the holo-reductase. 7. FAD, and adreno-ferredoxin and both could protect the apo-reductase from thermal inactivation.
Topics: Adrenal Cortex; Adrenodoxin; Animals; Apoenzymes; Cattle; Chemical Phenomena; Chemistry, Physical; Chromatography; Chromatography, Affinity; Ferredoxin-NADP Reductase; Flavin-Adenine Dinucleotide; Hot Temperature; Mitochondria; NADP; Protein Denaturation
PubMed: 2289620
DOI: 10.1016/0020-711x(90)90113-h -
Biochemical and Biophysical Research... May 2021Glucose-6-phosphate dehydrogenase is the first enzyme in the pentose phosphate pathway. The reaction catalyzed by the enzyme is considered to be the main source of...
Glucose-6-phosphate dehydrogenase is the first enzyme in the pentose phosphate pathway. The reaction catalyzed by the enzyme is considered to be the main source of reducing power for nicotinamide adenine dinucleotide phosphate (NADPH) and is a precursor of 5-carbon sugar used by cells. To uncover the structural features of the enzyme, we determined the crystal structures of glucose-6-phosphate dehydrogenase from Kluyveromyces lactis (KlG6PD) in both the apo form and a binary complex with its substrate glucose-6-phosphate. KlG6PD contains a Rossman-like domain for cofactor NADPH binding; it also presents a typical antiparallel β sheet at the C-terminal domain with relatively the same pattern as those of other homologous structures. Moreover, our structural and biochemical analyses revealed that Lys153 contributes significantly to substrate G6P recognition. This study may provide insights into the structural variation and catalytic features of the G6PD enzyme.
Topics: Amino Acid Sequence; Apoenzymes; Binding Sites; Catalytic Domain; Crystallography, X-Ray; Glucosephosphate Dehydrogenase; Kinetics; Kluyveromyces; Models, Molecular; Mutagenesis; Structure-Activity Relationship; Substrate Specificity
PubMed: 33765558
DOI: 10.1016/j.bbrc.2021.02.088 -
Journal of Bioscience and Bioengineering Jul 2015D-amino acid aminotransferase (D-AAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme. D-AAT from thermophilic Bacillus sp. YM-1 was inactivated by ultraviolet...
D-amino acid aminotransferase (D-AAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme. D-AAT from thermophilic Bacillus sp. YM-1 was inactivated by ultraviolet irradiation. The activity was restored by the addition of PLP and showed a linear correlation with respect to PLP concentrations in the range of 10-40 nM.
Topics: Amino Acids; Apoenzymes; Bacillus; Enzyme Assays; Pyridoxal Phosphate; Transaminases
PubMed: 25622769
DOI: 10.1016/j.jbiosc.2014.12.006 -
The FEBS Journal Sep 2015[Fe]-hydrogenase (Hmd), an enzyme of the methanogenic energy metabolism, harbors an iron-guanylylpyridinol (FeGP) cofactor used for H2 cleavage. The generated hydride is...
UNLABELLED
[Fe]-hydrogenase (Hmd), an enzyme of the methanogenic energy metabolism, harbors an iron-guanylylpyridinol (FeGP) cofactor used for H2 cleavage. The generated hydride is transferred to methenyl-tetrahydromethanopterin (methenyl-H4MPT(+)). Most hydrogenotrophic methanogens contain the hmd-related genes hmdII and hmdIII. Their function is still elusive. We were able to reconstitute the HmdII holoenzyme of Methanocaldococcus jannaschii with recombinantly produced apoenzyme and the FeGP cofactor, which is a prerequisite for in vitro functional analysis. Infrared spectroscopic and X-ray structural data clearly indicated binding of the FeGP cofactor. Methylene-H4MPT binding was detectable in the significantly altered infrared spectra of the HmdII holoenzyme and in the HmdII apoenzyme-methylene-H4 MPT complex structure. The related binding mode of the FeGP cofactor and methenyl-H4MPT(+) compared with Hmd and their multiple contacts to the polypeptide highly suggest a biological role in HmdII. However, holo-HmdII did not catalyze the Hmd reaction, not even in a single turnover process, as demonstrated by kinetic measurements. The found inactivity can be rationalized by an increased contact area between the C- and N-terminal folding units in HmdII compared with in Hmd, which impairs the catalytically necessary open-to-close transition, and by an exchange of a crucial histidine to a tyrosine. Mainly based on the presented data, a function of HmdII as Hmd isoenzyme, H2 sensor, FeGP-cofactor storage protein and scaffold protein for FeGP-cofactor biosynthesis could be excluded. Inspired by the recently found binding of HmdII to aminoacyl-tRNA synthetases and tRNA, we tentatively consider HmdII as a regulatory protein for protein synthesis that senses the intracellular methylene-H4 MPT concentration.
DATABASE
Structural data are available in the Protein Data Bank under the accession numbers 4YT8; 4YT2; 4YT4 and 4YT5.
Topics: Apoenzymes; Archaeal Proteins; Binding Sites; Coenzymes; Crystallography, X-Ray; Escherichia coli; Gene Expression; Holoenzymes; Hydrogenase; Iron-Sulfur Proteins; Kinetics; Methanocaldococcus; Models, Molecular; Protein Binding; Protein Biosynthesis; Protein Folding; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Pterins; Pyridines; Recombinant Proteins; Spectrophotometry, Infrared
PubMed: 26094576
DOI: 10.1111/febs.13351 -
The Journal of Biological Chemistry Aug 1975The interaction of a soluble homogeneous preparation of D-beta-hydroxybutyrate apodehydrogenase with phospholipid was studied in terms of restoration of enzymic activity...
The interaction of a soluble homogeneous preparation of D-beta-hydroxybutyrate apodehydrogenase with phospholipid was studied in terms of restoration of enzymic activity and complex formation. The purified apoenzyme, which is devoid of lipid, is inactive. It is reactivated specifically by the addition of lecithin or mixtures of phospholipids containing lecithin. Mitochondrial phospholipid, i.e. the mixture of phospholipids in mitochondria, reactivates with the highest specific activity (approximately 100 micromol of DPN reduced/min/mg at 37 degrees and with the greatest efficiency (2.5 to 4 mol of lecithin/mol of enzyme subunit). Each of the lecithins of varying chain length and unsaturation reactivated the enzyme, albeit to differing extents and efficiencies. In general, lecithins containing unsaturated fatty acid moieties reactivated better than those containing the comparable saturated lipid. Optimal reactivation can be obtained for the various lecithins when they are microdispersed together with phosphatidylethanolamine. When the lecithins are added microdispersed together with both phosphatidylethanolamine and cardiolipin, maximal efficiency is obtained. Also, PC6:0 and 8:0 reactivate as soluble molecules, so that a phospholipid bilayer is not necessary to reactivate the enzyme. Complex formation was studied using gel exclusion chromatography. It can be shown that each of the phospholipids which reactivate combines with the apoenzyme. Mitochondrial phospholipid, which reactivates the best, binds most effectively; PC8:0, which reactivates with poor efficiency, can be shown to bind with low affinity, and negligible binding occurs at concentrations which do not reactivate the enzyme. Since the apoenzyme is apparently homogeneous and devoid of phospholipid or detergents, it would appear that reactivation does not involve reversal of inhibition such as by removal of a regulatory subunit or detergent from the catalytic subunit. Rather, we conclude that phospholipid is a necessary and integral portion of this enzyme whose active form is a phospholipid-protein complex. The apoenzyme also forms a complex with phosphatidylethanolamine and/or cardiolipin, which do not reactivate enzymic activity. Salt dissociates such complexes in contrast with the lecithin-apoenzyme complex. Binding of phospholipid is a necessary but not sufficient requisite for enzymic activity. The same energies of activation are obtained from Arrhenius plots for the membrane-bound enzyme and for the purified soluble enzyme reactivated with mitochondrial phospholipid or different lecithins. This observation is compatible with the view that the purified enzyme has not been adversely modified in the isolation. Furthermore, essentially the same energies of activation were obtained for saturated lecithins below their transition temperatures and for unsaturated lecithins above their transition temperatures. Hence, there is no indication that a lipid phase transition occurs to influence the activity of this enzyme.
Topics: Animals; Apoenzymes; Apoproteins; Binding Sites; Cattle; Chromatography, Gel; Chromatography, Thin Layer; Fatty Acids; Fatty Acids, Unsaturated; Hydroxybutyrate Dehydrogenase; Kinetics; Mitochondria, Muscle; Myocardium; Phosphatidylcholines; Protein Binding; Structure-Activity Relationship; Temperature; Thermodynamics
PubMed: 1171100
DOI: No ID Found -
The Protein Journal Jun 2017Human ornithine δ-aminotransferase (hOAT) (EC 2.6.1.13) is a mitochondrial pyridoxal 5'-phosphate (PLP)-dependent aminotransferase whose deficit is associated with...
Human ornithine δ-aminotransferase (hOAT) (EC 2.6.1.13) is a mitochondrial pyridoxal 5'-phosphate (PLP)-dependent aminotransferase whose deficit is associated with gyrate atrophy, a rare autosomal recessive disorder causing progressive blindness and chorioretinal degeneration. Here, both the apo- and holo-form of recombinant hOAT were characterized by means of spectroscopic, kinetic, chromatographic and computational techniques. The results indicate that apo and holo-hOAT (a) show a similar tertiary structure, even if apo displays a more pronounced exposure of hydrophobic patches, (b) exhibit a tetrameric structure with a tetramer-dimer equilibrium dissociation constant about fivefold higher for the apoform with respect to the holoform, and (c) have apparent T values of 46 and 67 °C, respectively. Moreover, unlike holo-hOAT, apo-hOAT is prone to unfolding and aggregation under physiological conditions. We also identified Arg217 as an important hot-spot at the dimer-dimer interface of hOAT and demonstrated that the artificial dimeric variant R217A exhibits spectroscopic properties, T values and catalytic features similar to those of the tetrameric species. This finding indicates that the catalytic unit of hOAT is the dimer. However, under physiological conditions the apo-tetramer is slightly less prone to unfolding and aggregation than the apo-dimer. The possible implications of the data for the intracellular stability and regulation of hOAT are discussed.
Topics: Amino Acid Substitution; Apoenzymes; Enzyme Stability; Holoenzymes; Hot Temperature; Humans; Mutation, Missense; Ornithine-Oxo-Acid Transaminase; Protein Multimerization; Protein Structure, Quaternary
PubMed: 28345116
DOI: 10.1007/s10930-017-9710-5 -
Methods in Enzymology 1982
Topics: Anaerobiosis; Animals; Apoenzymes; Apoproteins; Humans; Prostaglandin-Endoperoxide Synthases; Prostaglandins H; Sheep
PubMed: 6813651
DOI: 10.1016/0076-6879(82)86170-3 -
Journal of Biochemistry Feb 1995The unfolding and refolding characteristics of Escherichia coli tryptophanase (tryptophan indole-lyase) [EC 4.1.99.1] in guanidine hydrochloride were studied....
The unfolding and refolding characteristics of Escherichia coli tryptophanase (tryptophan indole-lyase) [EC 4.1.99.1] in guanidine hydrochloride were studied. Tryptophanase unfolded by first dissociating its coenzyme, pyridoxal 5'-phosphate, from the active site. This dissociation caused a significant destabilization of structure, and global unfolding of the protein followed. During this global unfolding step, an intermediate was formed which had a strong tendency to aggregate irreversibly, as detected by light scattering experiments. Tryptophanase was unable to refold quantitatively after unfolding in 4 M guanidine hydrochloride. The low refolding yield was due to non-specific aggregation which occurs during refolding. Various conditions which limited this aggregation were probed, and it was found that by initiating the refolding reaction at low temperature, the aggregation of tryptophanase folding intermediates during the reaction could be avoided to a certain extent, and the refolding yield improved.
Topics: Apoenzymes; Escherichia coli; Guanidine; Guanidines; Indoleamine-Pyrrole 2,3,-Dioxygenase; Kinetics; Light; Protein Denaturation; Protein Folding; Pyridoxal Phosphate; Scattering, Radiation; Spectrometry, Fluorescence; Time Factors; Tryptophanase
PubMed: 7608129
DOI: 10.1093/jb/117.2.384 -
European Journal of Biochemistry Apr 1983The binding of 64Cu to the water-soluble form of dopamine beta-monooxygenase from bovine adrenal medulla was studied in reconstitution and exchange experiments using...
The binding of 64Cu to the water-soluble form of dopamine beta-monooxygenase from bovine adrenal medulla was studied in reconstitution and exchange experiments using high-performance size-exclusion gel chromatography. The reconstitution experiments provide evidence for a specific binding of four copper atoms/enzyme tetramer using either Cu(I) or Cu(II), but some weaker copper-binding sites were observed in the presence of a large excess of copper. The exchanges of both Cu(I) and Cu(II) in this protein are so rapid that exact half-lives for the exchange reactions can not be obtained by the present method. The results indicate, however, that the half-life for the exchange of the enzyme-bound copper in the holoenzyme with a twofold excess of 64Cu(II) at pH 6.1 was about 1 min, whereas the exchange of Cu(I) measured at similar conditions with ascorbate present, was complete in 1 min. This is by far the most rapid exchange reported for any copper-protein, and the results points to a unique copper-binding site in this enzyme.
Topics: Adrenal Medulla; Animals; Apoenzymes; Apoproteins; Binding Sites; Cattle; Chromatography, Gel; Chromatography, High Pressure Liquid; Copper; Dopamine beta-Hydroxylase; Protein Binding
PubMed: 6840081
DOI: 10.1111/j.1432-1033.1983.tb07343.x