Biochemistry (Moscow) (v.76, #6)

Structural-functional features of plant isoperoxidases by I. V. Maksimov; E. A. Cherepanova; G. F. Burkhanova; A. V. Sorokan’; O. I. Kuzmina (609-621).
Current data on structural-functional features of plant peroxidases and their involvement in functioning of the pro-/antioxidant system responding to stress factors, especially those of biotic origin, are analyzed. The collection of specific features of individual isoforms allows a plant to withstand an aggressive influence of the environment. Expression of some genes encoding different isoperoxidases is regulated by pathogens (and their metabolites), elicitors, and hormone-like compounds; specific features of this regulation are considered in detail. It is suggested that isoperoxidases interacting with polysaccharides are responsible for a directed deposition of lignin on the cell walls, and this lignin in turn is concurrently an efficient strengthening material and protects the plants against pathogens.
Keywords: peroxidases; genes; expression; structural-functional features; pathogens; resistance

This review deals with structural and functional features of glycoside hydrolases, a widespread group of enzymes present in almost all living organisms. Their catalytic domains are grouped into 120 amino acid sequence-based families in the international classification of the carbohydrate-active enzymes (CAZy database). At a higher hierarchical level some of these families are combined in 14 clans. Enzymes of the same clan have common evolutionary origin of their genes and share the most important functional characteristics such as composition of the active center, anomeric configuration of cleaved glycosidic bonds, and molecular mechanism of the catalyzed reaction (either inverting, or retaining). There are now extensive data in the literature concerning the relationship between glycoside hydrolase families belonging to different clans and/or included in none of them, as well as information on phylogenetic protein relationship within particular families. Summarizing these data allows us to propose a multilevel hierarchical classification of glycoside hydrolases and their homologs. It is shown that almost the whole variety of the enzyme catalytic domains can be brought into six main folds, large groups of proteins having the same three-dimensional structure and the supposed common evolutionary origin.
Keywords: glycoside hydrolase; CAZy; protein hierarchical classification; protein evolution; protein family; clan; superfamily; subfamily; search for homologs; TIM-barrel; SCOP; Pfam; PSI-BLAST

Role of lipid components in formation and reactivation of Mycobacterium smegmatis “nonculturable” cells by E. V. Nazarova; M. O. Shleeva; N. S. Morozova; Yu. K. Kudykina; G. N. Vostroknutova; A. O. Ruzhitsky; A. A. Selishcheva; G. M. Sorokoumova; V. I. Shvets; A. S. Kaprelyants (636-644).
We have found that transition of actively dividing Mycobacterium smegmatis cells into the dormant “nonculturable” state is accompanied by increase in the protein/lipid ratio and disappearance of one of the main lipid components of the mycobacterial cells, trehalose monomycolate. In this case, oleic acid is accumulated in the culture medium due to its secretion by the mycobacterial cells. Addition of lipids of different classes to “nonculturable” M. smegmatis cells induces their resuscitation. The lipid reactivating effect is evidently caused by the presence of fatty acids in their composition, because free fatty acids also exhibited reactivation effect. Oleic acid in concentration of 0.05–3 μg/ml exhibited maximal effect, and that allows us to draw a conclusion concerning its signal role in the transition of dormant cells into active state.
Keywords: mycobacteria; “nonculturable” state; oleic acid; lipids; reactivation

Rice dehydrin K-segments have in vitro antibacterial activity by C. Zhai; J. Lan; H. Wang; L. Li; X. Cheng; G. Liu (645-650).
Dehydrins are groups of plant proteins that have been shown to response to various environmental stimuli such as dehydration, elevated salinity, and low temperature. However, their roles in plant defense against microbes have not been demonstrated. In an attempt to discover plant antimicrobial proteins, we have screened a rice cDNA library and isolated several cDNAs coding for dehydrins. Protein extracts from Escherichia coli expressing these cDNAs were tested for their activity against Gram-positive bacteria (Bacillus pumilus, B. subtilis, Staphylococcus aureus, and Sarcina lutea) and Gramnegative bacteria (Escherichia coli and Xanthomonas oryzae pv. oryzae). The results indicate that the crude protein extracts exhibited antibacterial activities against the Gram-positive bacteria. However, dehydrins purified by immunoaffinity chromatography were not active against the bacteria. To pinpoint the dehydrin peptides that were responsible for the bactericidal activity, we expressed DNA sequences coding for truncated dehydrins containing either K- or S-segment and found that K-segment peptides, and not S-segment, were responsible for the antibacterial activities against Gram-positive bacteria. Antibacterial assay with synthetic K-segments indicated that the peptides inhibited growth of B. pumilus with minimum inhibition concentration and minimum bactericidal concentration of 130 and 400 μg/ml, respectively.
Keywords: dehydrin; antimicrobial peptide; antibacterial activity; K-segment; rice

Endonuclease WEN1 with apparent molecular mass about 27 kDa isolated from cytoplasmic vesicular fraction of aging coleoptiles of wheat seedlings has expressed site specificity action. This is a first detection and isolation of a site-specific endonuclease from higher eukaryotes, in general, and higher plants, in particular. The enzyme hydrolyzes deoxyri-booligonucleotides of different composition on CNG (N is G, A, C, or T) sites by splitting the phosphodiester bond between C and N nucleotide residues in CNG sequence independent from neighbor nucleotide context except for CCCG. WEN1 prefers to hydrolyze methylated λ phage DNA and double-stranded deoxyribooligonucleotides containing 5-methylcytosine sites (m5CAG, m5CTG) compared with unmethylated substrates. The enzyme is also able to hydrolyze single-stranded substrates, but in this case it splits unmethylated substrates predominantly. Detection in wheat seedlings of WEN1 endonuclease that is site specific, sensitive to the substrate methylation status, and modulated with S-adenosyl-L-methionine indicates that in higher plants restriction-modification systems or some of their elements, at least, may exist.
Keywords: 5-methylcytosine; AdoMet; CNG site; evolution; mitochondria; plant endonuclease; site specific action; wheat

Mycobacterium tuberculosis Rv0054 encodes a single-stranded DNA-binding protein (MtbSSB) that is essential for survival of the human pathogen and causative agent of tuberculosis. The function of MtbSSB has been proposed to be different from its E. coli homolog. However, the critical amino acid residues of MtbSSB and their regulatory effects on DNA-binding ability remain to be clearly characterized. In this study, using a frequency-controlled random mutagenesis method (FRM), mutant libraries of MtbSSB were successfully constructed. On the whole, 146 single, double, and triple MtbSSB mutants, which covered 89% of the amino acid residues along the whole MtbSSB gene, were isolated. Using bacterial two-hybrid assays in combination with native PAGE assays, four new mutants, E62G, D104N, E94G/T137N, and S130P/G153N were found to totally or partially lose their ability to form tetramer. Three novel mutants, E62G, D104N, and E94G/T134N, were characterized to have a much lower ssDNA-binding activity, while one mutant, F21L, was found to have a significantly higher activity through both electrophoretic mobility shift and surface plasmon resonance assays. Interestingly, three amino acid residues, E62, D104, and E94, were found to regulate both oligomerization and ssDNA-binding activity of MtbSSB. Our work provides an important resource and should help improve the understanding of the biochemical mechanisms and structure-function relationship of the DNA-binding protein in this important human pathogen.
Keywords: Mycobacterium tuberculosis ; single-stranded DNA-binding protein; random mutagenesis; GC-rich gene

Expression, purification, and characterization of putative Candida albicans Rad3, the product of orf19.7119 by Ki Moon Seong; Se Hyun Lee; Hag Dong Kim; Chang Hoon Lee; Hyesook Youn; BuHyun Youn; Joon Kim (666-676).
Invasive infections of Candida albicans are life-threatening clinical conditions affecting immunosuppressed patients. To maintain genome integrity and diversity, C. albicans utilizes DNA repair systems, such as nucleotide excision repair (NER), to escape from attack by macrophages. Rad3 helicase is a component of the TFIIH complex, which plays a role in transcription and the NER pathway. Accumulated evidence of studies from Archaea to humans has revealed that the conserved structure, including an iron-containing domain, is essential in the function of Rad3 helicase activity. However, no study of the Rad3 protein of C. albicans has yet been reported. In the present study, putative C. albicans Rad3 (CaRad3) has been cloned with orf19.7119 of the Candida genome. CaRad3 proteins were over-expressed and purified from E. coli and S. cerevisiae using a Ni-NTA column and a size exclusion column for physicochemical and functional characterization. Through EMR and spectrometric analysis, we have proven that the purified CaRad3 protein has a Fe-S cluster. We also revealed that CaRad3 protein has a helicase activity on a duplex DNA substrate. Furthermore, we showed that the CaRad3 protein purified from yeasts was N-glycosylated, and that this protein complemented the defects in both the NER pathway and transcription. These data suggest that the Rad3 helicase in C. albicans is the product of the orf19.7119 gene.
Keywords: Candida albicans ; Rad3; DNA repair; Fe-S cluster; functional complementation

Free radical reaction products and antioxidant capacity in beating heart coronary artery surgery compared to conventional bypass by A. Gonenc; A. Hacışevki; H. R. Griffiths; M. Torun; B. Bakkaloglu; B. Simsek (677-685).
Oxygen-derived free radicals are important agents of tissue injury during ischemia and reperfusion. The aim of this study was to investigate changes in protein and lipid oxidation and antioxidant status in beating heart coronary artery surgery and conventional bypass and to compare oxidative stress parameters between the two bypass methods. Serum lipid hydroperoxide, nitric oxide, protein carbonyl, nitrotyrosine, vitamin E, and β-carotene levels and total antioxidant capacity were measured in blood of 30 patients undergoing beating heart coronary artery surgery (OPCAB, off-pump coronary artery bypass grafting) and 12 patients undergoing conventional bypass (CABG, on-pump coronary artery bypass grafting). In the OPCAB group, nitric oxide and nitrotyrosine levels decreased after reperfusion. Similarly, β-carotene level and total antioxidant capacity also decreased after anesthesia and reperfusion. In the CABG group, nitric oxide and nitrotyrosine levels decreased after ischemia and reperfusion. However, protein carbonyl levels elevated after ischemia and reperfusion. Vitamin E, β-carotene, and total antioxidant capacity decreased after ischemia and reperfusion. Significantly decreased nitration and impaired antioxidant status were seen after reperfusion in both groups. Moreover, elevated protein carbonyls were found in the CABG group. The off-pump procedure is associated with lower degree of oxidative stress than on-pump coronary surgery.
Keywords: oxidative stress; coronary artery bypass; antioxidant; protein carbonyls; nitrotyrosine

A novel protease from the halophilic bacterium Geomicrobium sp. EMB2 (MTCC 10310) is described. The activity of the protease was modulated by salt, and it exhibited remarkable stability in organic solvents, at alkaline pH, and in other denaturing conditions. The structural changes under various denaturing conditions were analyzed by measurements of intrinsic fluorescence and circular dichroism spectroscopy. Circular dichroism showed that the secondary structure of the protease was predominantly α-helical but unfolded in salt-free medium. The structure is regained by inclusion of NaCl in the range of 2–5%. The presence of NaCl exerted a protective effect against thermal, organic solvent, and guanidine hydrochloride denaturation by preventing unfolding.
Keywords: halophiles; solvent stability; protein folding; protease; circular dichroism; fluorescence

Purification, primary structure, and properties of Euphorbia characias latex purple acid phosphatase by F. Pintus; D. Spano; S. Corongiu; G. Floris; R. Medda (694-701).
A purple acid phosphatase was purified to homogeneity from Euphorbia characias latex. The native protein has a molecular mass of 130 ± 10 kDa and is formed by two apparently identical subunits, each containing one Fe(III) and one Zn(II) ion. The two subunits are connected by a disulfide bridge. The enzyme has an absorbance maximum at 540 nm, conferring a characteristic purple color due to a charge-transfer transition caused by a tyrosine residue (Tyr172) coordinated to the ferric ion. The cDNA nucleotide sequence contains an open reading frame of 1392 bp, and the deduced sequence of 463 amino acids shares a very high degree of identity (92–99%) to other purple acid phosphatases isolated from several higher plants. The enzyme hydrolyzes well p-nitrophenyl phosphate, a typical artificial substrate, and a broad range of natural phosphorylated substrates, such as ATP, ADP, glucose-6-phosphate, and phosphoenolpyruvate. The enzyme displays a pH optimum of 5.75 and is inhibited by molybdate, vanadate, and Zn2+, which are typical acid phosphatase inhibitors.
Keywords: acid phosphatase; Euphorbia characias ; iron ion; metalloenzymes; purple phosphatase; zinc ion

Since modern Europeans appear to be descendants of the Late Pleistocene European peoples who survived the last glacial period, it is quite reasonable to expect the presence of adaptive genetic variants that originated in the Ice Age in the modern gene pool of Europeans. To find such adaptive variants, mitochondrial genomes have been analyzed of the modern population from Eastern and Central Europe belonging to haplogroups U4, U5, and V, that diversified during the Late Pleistocene and Holocene periods. Analysis of distribution of nonsynonymous and synonymous substitutions, as well as results of search for radical amino acid changes that arose under the influence of adaptation (positive destabilizing selection) allowed us to detect signals of molecular adaptation in different mitochondrial genes and haplogroups of mtDNA. However, there were very few strong adaptive signals (z > 3.09, P < 0.001) that could be due to the loss of adaptive mtDNA haplotypes during the Holocene warming.
Keywords: human mitochondrial genome; adaptive evolution; selection

Structure of the O-polysaccharide of Providencia alcalifaciens O25 containing an amide of D-galacturonic acid with N ɛ-[(R)-1-carboxyethyl]-L-lysine by N. A. Kocharova; O. G. Ovchinnikova; M. Bialczak-Kokot; A. S. Shashkov; Y. A. Knirel; A. Rozalski (707-712).
An acidic O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O25 followed by gel-permeation and anion-exchange chromatography. The O-polysaccharide was studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including two-dimensional correlation 1H,13C HMBC, and 1H,1H ROESY experiments both in D2O and, to detect correlations for NH protons, in a 9: 1 H2O/D2O mixture. An amino acid was isolated from the polysaccharide by acid hydrolysis and identified as N ɛ-[(R)-1-carboxyethyl]-L-lysine (“alaninolysine”, 2S,8R-alaLys) by determination of the specific optical rotation and 13C NMR spectroscopy, using the authentic synthetic diastereomers 2S,8R-alaLys and 2S,8S-alaLys for comparison. The structure of the branched tetrasaccharide repeating unit of the O-polysaccharide was established.
Keywords: Providencia alcalifaciens ; O-antigen; lipopolysaccharide; bacterial polysaccharide structure; opine; N ɛ-[(R)-1-carboxyethyl]-L-lysine

Recombinant polyamine-binding protein of Synechocystis sp. PCC 6803 specifically binds to and is induced by polyamines by P. Yodsang; W. Raksajit; A. -M. Brandt; T. A. Salminen; P. Mäenpää; A. Incharoensakdi (713-719).
His-tagged Synechocystis sp. PCC 6803 PotD protein (rPotD) involved in polyamine transport was overexpressed in Escherichia coli. The purified rPotD showed saturable binding kinetics with radioactively labeled polyamines. The rPotD exhibited a similar binding characteristic for three polyamines, with putrescine having less preference. The K d values for putrescine, spermine, and spermidine were 13.2, 8.3, and 7.8 μM, respectively. Binding of rPotD with polyamines was maximal at pH 8.0. Docking of these polyamines into the homology model of Synechocystis PotD showed that all three polyamines are able to interact with Synechocystis PotD. The binding modes of the docked putrescine and spermidine in Synechocystis are similar to those of PotF and PotD in E. coli, respectively. Competition experiments showed specific binding of rPotD with polyamines. The presence of putrescine and spermidine in the growth medium could induce an increase in PotD contents, suggesting the role of PotD in mediating the transport of polyamine in Synechocystis sp. PCC 6803.
Keywords: polyamine binding; polyamine docking; PotD induction; rPotD; Synechocystis sp. PCC 6803

New approaches to identification and activity estimation of glyphosate degradation enzymes by A. V. Sviridov; N. F. Zelenkova; N. G. Vinokurova; I. T. Ermakova; A. A. Leontievsky (720-725).
We propose a new set of approaches, which allow identifying the primary enzymes of glyphosate (N-phosphonomethyl-glycine) attack, measuring their activities, and quantitative analysis of glyphosate degradation in vivo and in vitro. Using the developed approach we show that glyphosate degradation can follow different pathways depending on physiological characteristics of metabolizing strains: in Ochrobactrum anthropi GPK3 the initial cleavage reaction is catalyzed by glyphosate-oxidoreductase with the formation of aminomethylphosphonic acid and glyoxylate, whereas Achromobacter sp. MPS12 utilize C-P lyase, forming sarcosine. The proposed methodology has several advantages as compared to others described in the literature.
Keywords: glyphosate; liquid chromatography; thin layer chromatography; C-P lyase; glyphosate-oxidoreductase

Erratum to: “Effect of Plastoquinone Derivative 10-(6′-Plastoquinonyl)decyltriphenylphosphonium (SkQ1) on Contents of Steroid Hormones and NO Level in Rats” by V. A. Chistyakov; V. A. Serezhenkov; A. A. Alexandrova; N. P. Milyutina; V. N. Prokof’ev; E. V. Mashkina; L. V. Gutnikova; S. V. Dem’yanenko (726-726).