Biochemistry (Moscow) (v.75, #10)

Proteinaceous inhibitors of microbial xylanases by A. V. Gusakov (1185-1199).
At the end of 1990s two structurally different proteinaceous inhibitors of xylanases were discovered in the grain of wheat (Triticum aestivum). They were named TAXI (T. aestivum xylanase inhibitor) and XIP (xylanase-inhibiting protein). Later it was shown that TAXI and XIP in wheat are present in several isoforms encoded by different genes. TAXI- and XIP-like inhibitors have also been found in other cereals-barley, rye, rice, maize, etc. All these proteins can specifically inhibit activity of fungal and bacterial xylanases belonging to families 10 and 11 of glycoside hydrolases, but they do not affect endogenous enzymes produced by plants. A common viewpoint is that the presence of proteinaceous inhibitors in cereals is a response of plants to pathogenic attack by microorganisms. A few years ago, an inhibitor of a third type was discovered in wheat. It was named TLXI (thaumatin-like xylanase inhibitor) because of its similarity to the thaumatin family of plant proteins. In this review, the occurrence of proteinaceous inhibitors of xylanases in different cereals, their specificity towards fungal and bacterial enzymes, as well as structural features responsible for enzyme sensitivity to various types of inhibitors are discussed.
Keywords: xylanase; cereals; inhibition; TAXI; XIP; TLXI

Excited flavin and pterin coenzyme molecules in evolution by M. S. Kritsky; T. A. Telegina; Y. L. Vechtomova; M. P. Kolesnikov; T. A. Lyudnikova; O. A. Golub (1200-1216).
Excited flavin and pterin molecules are active in intermolecular energy transfer and in photocatalysis of redox reactions resulting in conservation of free energy. Flavin-containing pigments produced in models of the prebiotic environment are capable of converting photon energy into the energy of phosphoanhydride bonds of ATP. However, during evolution photochemical reactions involving excited FMN or FAD molecules failed to become participants of bioenergy transfer systems, but they appear in enzymes responsible for repair of UV-damaged DNA (DNA photolyases) and also in receptors of blue and UV-A light regulating vital functions of organisms. The families of these photoproteins (DNA-photolyases and cryptochromes, LOV-domain- and BLUF-domain-containing proteins) are different in the structure and in mechanisms of the photoprocesses. The excited flavin molecules are involved in photochemical processes in reaction centers of these photoproteins. In DNA photolyases and cryptochromes the excitation energy on the reaction center flavin is supplied from an antenna molecule that is bound with the same polypeptide. The role of antenna is played by MTHF or by 8-HDF in some DNA photolyases, i.e. also by molecules with known coenzyme functions in biocatalysis. Differences in the structure of chromophore-binding domains suggest an independent origin of the photoprotein families. The analysis of structure and properties of coenzyme molecules reveals some specific features that were significant in evolution for their being selected as chromophores in these proteins.
Keywords: evolution; photoreceptors; photocatalysis; excited states; selection of chromophores; coenzymes; flavins; pterins; folates; DNA photolyases; cryptochromes; phototropins; LOV domain; BLUF domain; modeling of prebiotic processes; photophosphorylation

Bacterial NO synthases by S. Yu. Filippovich (1217-1224).
Unlike mammalian NO synthases, bacterial NO synthases do not contain a reductase domain. The only exception from this rule is the NO synthase from myxobacterium Sorangium cellulosum, but its reductase domain has unusual structure and location in the enzyme molecule. Recent achievements in bacterial genome sequencing have revealed the gene coding NO synthase (represented as an oxygenase domain) in some bacteria and have advanced the study of structure and functions of bacterial NO synthases. Important features of structure, sources of reducing equivalents, evolutionary connections, and functions of bacterial NO synthases (i.e. participation in nitration of the indole ring of Trp, in reparation of UV-radiation damage, role in adaptation of bacteria to oxidative stress, participation in the synthesis of cGMP, and resistance of bacteria against antibiotics) are described.
Keywords: NO synthase; nitric oxide; oxygenase domain; bacteria

Conformational dynamics and pre-steady-state kinetics of DNA glycosylases by O. S. Fedorova; N. A. Kuznetsov; V. V. Koval; D. G. Knorre (1225-1239).
Results of investigations of E. coli DNA glycosylases using pre-steady-state kinetics are considered. Special attention is given to the connection of conformational changes in the interacting biomolecules with kinetic mechanisms of the enzymatic processes.
Keywords: conformational dynamics; pre-steady-state kinetics; DNA glycosylases

Proteins tightly bound to DNA: New data and old problems by N. Sjakste; L. Bagdoniene; A. Gutcaits; D. Labeikyte; K. Bielskiene; I. Trapina; I. Muiznieks; Y. Vassetzky; T. Sjakste (1240-1251).
Proteins tightly bound to DNA (TBP) comprise a group of proteins that remain bound to DNA after usual deproteinization procedures such as salting out and treatment with phenol or chloroform. TBP bind to DNA by covalent phosphotriester and noncovalent ionic and hydrogen bonds. Some TBP are conservative, and they are usually covalently bound to DNA. However, the TBP composition is very diverse and significantly different in different tissues and in different organisms. TBP include transcription factors, enzymes of the ubiquitin-proteasome system, phosphatases, protein kinases, serpins, and proteins of retrotransposons. Their distribution within the genome is nonrandom. However, the DNA primary structure or DNA curvatures do not define the affinity of TBP to DNA. But there are repetitive DNA sequences with which TBP interact more often. The TBP distribution within genes and chromosomes depends on a cell’s physiological state, differentiation type, and stage of organism development. TBP do not interact with DNA in the sites of its association with nuclear matrix and most likely they are not components of the latter.
Keywords: proteins tightly bound to DNA; nuclear matrix; repetitive DNA sequences; serpins; phosphatases; transcription factors; differentiation

Proteomic characterization of Mycoplasma gallisepticum nanoforming by I. A. Demina; M. V. Serebryakova; V. G. Ladygina; M. A. Rogova; I. G. Kondratov; A. N. Renteeva; V. M. Govorun (1252-1257).
The goal of this work was to create a model for the long persistence of Mycoplasma gallisepticum in depleted medium and under low growth temperature followed by proteomic study of the model. Nanoforms and revertants for M. gallisepticum were obtained. Proteomic maps were produced for different stages of the formation of nanoforms and revertants. It is shown that proteins responsible for essential cellular processes of glycolysis, translation elongation, and DnaK chaperone involved in the stabilization of newly synthesized proteins are crucial for the reversion of M. gallisepticum to a vegetative form. Based on the current data, it is assumed that changes in the metabolism of M. gallisepticum during nanoforming are not post-mortal, thus M. gallisepticum does not transform to uncultivable form, but remains in a reversible dormant state during prolonged unfavorable conditions.
Keywords: Mycoplasma gallisepticum ; adaptation; starvation; reversion; proteomics; 2-D electrophoresis; mass spectrometry; real-time PCR

Structures closed into cycles in proteins containing 3β-corners by E. A. Boshkova; A. V. Efimov (1258-1263).
In the present study, pathways of growth of protein structures represented in the structural tree for β-proteins containing 3β-corners are analyzed. It is shown that the frequency of occurrence of the completed structures of known proteins within branches of the tree is quite different. This means that allowed pathways of growth of protein structures are not equal and their usage is quite different. In most cases, addition of one or two β-strands nearest along the chain to the root 3β-corner (67%) or addition of three β-strands to the 3β-corner results in the formation of structures closed into cycles or barrels. Therefore, the pathways that result in closed structures are used most often in the first steps of growth of the root 3β-corner. Amino acid sequences coding for left-handed superhelices that close into cycles the 3β-corners are also analyzed. It is demonstrated that most crossover sites where the polypeptide chain passes from one β-layer to the other have one or two residues in sterically constrained αL- or ɛ-conformations, which should be glycines or residues with flexible side chains in order to reduce the steric constraints.
Keywords: protein structure modeling; protein folding; structural trees

Paracoccus denitrificans proton-translocating ATPase: Kinetics of oxidative phosphorylation by K. A. Kegyarikova; T. V. Zharova; A. D. Vinogradov (1264-1271).
The initial rates of ATP synthesis catalyzed by tightly coupled Paracoccus denitrificans plasma membrane were measured. The reaction rate was hyperbolically dependent on the substrates, ADP and inorganic phosphate (Pi). Apparent K m values for ADP and Pi were 7–11 and 60–120 μM, respectively, at saturating concentration of the second substrate (pH 8.0, saturating Mg2+). These values were dependent on coupling efficiency. The substrate binding in the ATP synthesis reaction proceeds randomly: K m value for a given substrate was independent of the concentration of the other one. A decrease of electrochemical proton gradient by the addition of malonate (when succinate served as the respiratory substrate) or by a decrease of steady-state level of NADH (when NADH served as the respiratory substrate) resulted in a proportional decrease of the maximal rates and apparent K m values for ADP and Pi (double substitution, ping-pong mechanism). The kinetic scheme for ATP synthesis was compared with that described previously for the proton-translocating ATP hydrolysis catalyzed by the same enzyme preparation (T. V. Zharova and A. D. Vinogradov (2006) Biochemistry, 45, 14552–14558).
Keywords: FoF1-H+-ATP synthase; oxidative phosphorylation; bacterial plasma membrane vesicles; Paracoccus denitrificans

Kinetic and spectral parameters of interaction of Citrobacter freundii methionine γ-lyase with amino acids by E. A. Morozova; N. P. Bazhulina; N. V. Anufrieva; D. V. Mamaeva; Y. V. Tkachev; S. A. Streltsov; V. P. Timofeev; N. G. Faleev; T. V. Demidkina (1272-1280).
Kinetic parameters of Citrobacter freundii methionine γ-lyase were determined with substrates in γ-elimination reactions as well as the inhibition of the enzyme in the γ-elimination of L-methionine by amino acids with different structure. The data indicate an important contribution of the sulfur atom and methylene groups to the efficiency of binding of substrates and inhibitors. The rate constants of the enzyme-catalyzed exchange of C-α- and C-β-protons with deuterium were determined, as well as the kinetic isotope effect of the deuterium label in the C-α-position of inhibitors on the rate of exchange of their β-protons. Neither stereoselectivity in the β-proton exchange nor noticeable α-isotope effect on the exchange rates of β-protons was found. The ionic and tautomeric composition of the external Schiff base of methionine γ-lyase was determined. Spectral characteristics (absorption and circular dichroism spectra) of complexes with substrates and inhibitors were determined. The spectral and kinetic data indicate that deamination of aminocrotonate should be the ratedetermining stage of the enzymatic reaction.
Keywords: Citrobacter freundii methionine γ-lyase; pyridoxal 5′-phosphate; substrates; inhibitors; proton exchange; spectral characteristics

Neutral endopeptidase neprilysin is copurified with Na,K-ATPase from rabbit outer medulla and hydrolyzes its α-subunit by M. A. Groubman; Y. V. Kamanina; I. Yu. Petrushanko; A. M. Rubtsov; O. D. Lopina (1281-1284).
Preparations of Na,K-ATPase from outer medulla of rabbit kidney purified in accordance with the method of P. L. Jorgensen were shown to contain as admixture a protease that moves with α-subunit (∼100 kDa) as a single protein band during one-dimensional SDS-PAGE. The electro-elution of proteins of this band from polyacrylamide gel results in the appearance of two protein fragments (∼67 and 55 kDa) that are stained with polyclonal antibodies against Na,K-ATPase α-subunit. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis showed that the neutral membrane-bound endopeptidase neprilysin is located in one protein band together with the Na,K-ATPase α-subunit. Addition of thiorphan, a specific inhibitor of neutral endopeptidase, eliminates proteolysis of the α-subunit. The data demonstrate that Na,K-ATPase α-subunit may be a natural target for neprilysin.
Keywords: Na,K-ATPase α-subunit; neprilysin; proteolysis; mammalian kidney

Oxidized modification of fragments D and E from fibrinogen induced by ozone by M. A. Rosenfeld; V. B. Leonova; A. N. Shchegolikhin; S. D. Razumovskii; M. L. Konstantinova; A. V. Bychkova; A. L. Kovarskii (1285-1293).
Ozone-induced free-radical oxidation of fragments D and E from fibrinogen has been studied. The methods of elastic and dynamic light scattering in combination with electrophoresis of unreduced samples have shown the acceleration of enzymatic covalent crosslinking of molecules of oxidation-modified fragment D under the action of factor XIIIa. UV and IR spectroscopy shows that free-radical oxidation of amino acid residues of polypeptide chains catalyzed by ozone affects the cyclic and amino groups, giving rise to generation of mainly oxygen-containing products. Comparison of the IR spectra obtained for the oxidation-modified D and E fragments revealed more significant transformation of functional groups for the D fragment. EPR spectroscopy showed that the rotational correlation time of spin labels bound to the ozonized proteins decreased in comparison with the non-ozonized proteins. The rotation correlation time of the radicals covalently bound to the ozonized D and E fragments suggests that D fragment of fibrinogen is more sensitive to free-radical oxidation followed by local structural changes. Possible causes of different degrees of oxidation for fragments D and E are discussed.
Keywords: fibrinogen; fragments D and E ; factor XIIIa; oxidation; structure

Characteristics of a novel secreted zinc-dependent endopeptidase of Bacillus intermedius by N. L. Rudakova; N. P. Balaban; Y. V. Danilova; G. N. Rudenskaya; M. R. Sharipova (1294-1301).
A novel zinc-dependent metalloendopeptidase of Bacillus intermedius (MprBi) was purified from the culture medium of a recombinant strain of Bacillus subtilis. The amino acid sequence of the homogeneous protein was determined using MALDI-TOF mass spectrometry. The sequence of the first ten residues from the N-terminus of the mature protein is ASTGSQKVTV. Physicochemical properties of the enzyme and its substrate specificity have been studied. The molecular weight of the metalloproteinase constitutes 19 kDa, the K m and k cat values are 0.06 mM and 1210 sec−1, respectively, and the pI value is 5.4. The effect of different inhibitors and metal ions on the enzyme activity has been studied. Based on the analysis of the amino acid sequence of the active site motif and the Met-turn together with the enzyme characteristics, the novel bacterial metalloproteinase MprBi is identified as a metzincin clan adamalysin/reprolysin-like metalloprotease.
Keywords: metalloproteinase; astacins; adamalysins/reprolysins; metzincins; Bacillus subtilis ; Bacillus intermedius

Erratum: “Binding of ATP and Its Derivatives to Selenophosphate Synthetase from Escherichia coli by Y. V. Preabrazhenskaya; I. Y. Kim; T. C. Stadtman (1302-1302).

Erratum: “A Novel Low Molecular Weight Alanine Aminotransferase from Fasted Rat Liver” by M. Vedavathi; K. S. Girish; M. Karuna Kumar (1303-1303).

Erratum: “Isoforms of Human O-GlcNAcase Show Distinct Catalytic Efficiencies” by Jing Li; Cai-luan Huang; Lian-wen Zhang; Lin Lin; Zhong-hua Li; Fu-wu Zhang; Peng Wang (1305-1305).