Lili Kandra | University of Debrecen (original) (raw)

Papers by Lili Kandra

Biochemical and enzymatic characterisation has been achieved for the Tyr151Met (Y151M) mutant of ... more Biochemical and enzymatic characterisation has been achieved for the Tyr151Met (Y151M) mutant of human salivary α-amylase (HSA). Substantial transglycosylation capacity was detected in Y151M in addition to its hydrolytic activity. Y151M was capable of transferring maltose and maltotriose residues from a maltotetraose donor onto different 4-nitrophenyl glycosides resulting in the formation of 1-thio-β-D-glucosides, β-and α-D-glucosides and β-D-xylosides with DP 2-4 in yields up to 50%. Reactions were monitored using TLC, HPLC and MALDI-TOF MS. 1 H and 13 C NMR studies revealed that the Y151M preserved its stereo-and regio-selectivity. Glycosylation took place at position 4 of the glycosyl acceptors, resulting in the new α-1,4-glycosidic bonds exclusively.

A biológiai szabályozási folyamatok negatív töltésű szénhidrátjai uronsavakat, szulfátészter csop... more A biológiai szabályozási folyamatok negatív töltésű szénhidrátjai uronsavakat, szulfátészter csoportokat vagy N-acetil-neuraminsavat tartalmaznak. Célunk a cukorvázon szulfonsavat vagy metilénszulfonsavat hordozó analógok előállítása volt. Potenciálisan antiadhéziós és antimetaszatikus di- és triszacharid-szulfonsavakat szintetizáltunk anomer pozícióban szulfonometil elágazást tartalmazó tioglikozid-donorokkal. A tioglikozidokból glikozilezéskor lejátszódó eliminációs mellékreakció visszaszorítására fluorid-, klorid- és bromid-donorokat preparáltunk az 1-etoxiszulfonilmetil-heptulózokból és megvizsgáltuk glikozilezési tulajdonságaikat. A leghatékonyabbnak bizonyult glikozil kloridok felhasználásával cukoregységenként egy szulfonsavcsoportot tartalmazó, tetraszacharidig terjedő maltooligomer-típusú oligoszacharid sorozatokat állítottunk elő, esetükben heparin-analóg, és amiláz inhibitor hatás várható. L-Fukózból előállítottuk az N-acetilneuraminsav szulfonsav anlogonjait, neuraminidá...

Action pattern of α-amylases on modified maltooligosaccharides. Biologia, Bratislava, 57/Suppl. 1... more Action pattern of α-amylases on modified maltooligosaccharides. Biologia, Bratislava, 57/Suppl. 11: 171-180, 2002; ISSN 0006-3088.

Phytochemistry, Jan 20, 1998

Compounds volatilized from plant tissues play important roles in plant-insect and plant-herbivore... more Compounds volatilized from plant tissues play important roles in plant-insect and plant-herbivore interactions and are important to food quality/preference, and to the perfume and flavorant industries. While the chemistry of plant volatiles is well understood, less is known about the biosynthesis of this diverse group of compounds. This is particularly the case for non-terpenoid components such as volatile acyclic alcohols and their esters. Here we have studied metabolic pathways leading to formation of the anteiso-branched alcohol 4-methyl-1-hexanol volatilized by petal tissue of Nicotiana sylvestris. Evidence presented supports the involvement of steps in the pathways of both biosynthesis and degradation of isoleucine to form 2-oxo-3-methylvaleric acid then 2-methylbutyryl CoA. Results indicate that 2-methylbutyryl CoA is then elongated by addition of one acetate molecule via fatty acid synthase, followed by reduction to yield 4-methyl-1-hexanol. This pathway is in contrast to elo...

Journal of Molecular Structure: THEOCHEM, 2003

α-Amylases (α-1,4-glucan-4-glucanohydrolases; EC 3.2.1.1) are classical calcium-containing enzyme... more α-Amylases (α-1,4-glucan-4-glucanohydrolases; EC 3.2.1.1) are classical calcium-containing enzymes, which constitute a family of endo-amylases catalysing the cleavage of α-d-(1-4) glycosidic bonds in starch and related carbohydrates with retention of the α-anomeric configuration in the products. They can be found in microorganisms, plants and higher organisms where they play a dominant role in carbohydrate metabolism. This study characterizes the substrate binding

European Journal of Biochemistry, 1990

A major diversion of carbon from branched-chain amino acid biosynthesis/catabolism to form acyl m... more A major diversion of carbon from branched-chain amino acid biosynthesis/catabolism to form acyl moieties of sucrose esters (6-~-acety~-~,~,~-tr~-~-acy~-a-~-g~ucopyranosy~-~-~-fructofuranos~des) was observed to be associated with specialized trichome head cells which secrete large amounts of sucrose esters. Surface chemistry and acetyl and acyl substituent groups of tobacco (T.I. 1068) sucrose esters were identified and quantified by gas chromatography/mass spectrometry. Sucrose esters were prominent surface constituents and 3-methylvaleric acid, 2-and 3-methylbutyric acid, and methylpropionic acid accounted for 60%, 25% and 9%, respectively, of total C3 -C7 acyl substituents. Radiolabeled Thr, Ile, Val, Leu, pyruvate and Asp, metabolites of branched-chain amino acid pathways, were compared with radioactively labeled acetate and sucrose as donors of carbon to sucrose, acetyl and acyl components of sucrose esters using epidermal peels with undisturbed trichomes. Preparations of biosynthetically competent trichome heads (site of sucrose ester formation) were also examined. Results indicate that 3-methylvaleryl and 2-methylbutyryl groups are derived from the Thr pathway of branched-chain amino acid metabolism, 3-methylbutyryl and methylpropionyl groups are formed via the pyruvate pathway, and that acetyl groups are principally formed directly via acetyl-CoA. Arguments are presented which rule out participation of fatty acid synthase in the formation of prominent acyl acids.

European Journal of Biochemistry, 2004

The nonreducing end of the substrate-binding site of human salivary alpha-amylase contains two re... more The nonreducing end of the substrate-binding site of human salivary alpha-amylase contains two residues Trp58 and Trp59, which belong to beta2-alpha2 loop of the catalytic (beta/alpha)(8) barrel. While Trp59 stacks onto the substrate, the exact role of Trp58 is unknown. To investigate its role in enzyme activity the residue Trp58 was mutated to Ala, Leu or Tyr. Kinetic analysis of the wild-type and mutant enzymes was carried out with starch and oligosaccharides as substrates. All three mutants exhibited a reduction in specific activity (150-180-fold lower than the wild type) with starch as substrate. With oligosaccharides as substrates, a reduction in k(cat), an increase in K(m) and distinct differences in the cleavage pattern were observed for the mutants W58A and W58L compared with the wild type. Glucose was the smallest product generated by these two mutants in the hydrolysis oligosaccharides; in contrast, wild-type enzyme generated maltose as the smallest product. The production of glucose by W58L was confirmed from both reducing and nonreducing ends of CNP-labeled oligosaccharide substrates. The mutant W58L exhibited lower binding affinity at subsites -2, -3 and +2 and showed an increase in transglycosylation activity compared with the wild type. The lowered affinity at subsites -2 and -3 due to the mutation was also inferred from the electron density at these subsites in the structure of W58A in complex with acarbose-derived pseudooligosaccharide. Collectively, these results suggest that the residue Trp58 plays a critical role in substrate binding and hydrolytic activity of human salivary alpha-amylase.

European Journal of Biochemistry, 2002

A computer program has been evaluated for subsite map calculations of depolymerases. The program ... more A computer program has been evaluated for subsite map calculations of depolymerases. The program runs in windows and uses the experimentally determined bond cleavage frequencies (BCFs) for determination of the number of subsites, the position of the catalytic site and for calculation of subsite binding energies. The apparent free energy values were optimized by minimization of the differences of the measured and calculated BCF data. The program called suma (SUbsite Mapping of alpha-Amylases) is freely available for research and educational purposes via the Internet (E-mail: gyemant@tigris.klte.hu). The advantages of this program are demonstrated through alpha-amylases of different origin, e.g. porcine pancreatic alpha-amylase (PPA) studied in our laboratory, in addition to barley and rice alpha-amylases published in the literature. Results confirm the popular &amp;amp;amp;amp;amp;amp;amp;#39;five subsite model&amp;amp;amp;amp;amp;amp;amp;#39; for PPA with three glycone and two aglycone binding sites. Calculations for barley alpha-amylase justify the &amp;amp;amp;amp;amp;amp;amp;#39;6 + 2 + (1) model&amp;amp;amp;amp;amp;amp;amp;#39; prediction. The binding area of barley alpha-amylase is composed of six glycone, two aglycone binding sites followed by a barrier subsite at the reducing end of the binding site. Calculations for rice alpha-amylase represent an entirely new map with a &amp;amp;amp;amp;amp;amp;amp;#39;(1) + 2 + 5 model&amp;amp;amp;amp;amp;amp;amp;#39;, where &amp;amp;amp;amp;amp;amp;amp;#39;(1)&amp;amp;amp;amp;amp;amp;amp;#39; is a barrier subsite at the nonreducing end of the binding site and there are two glycone and five aglycone binding sites. The rice model may be reminiscent of the action of the bacterial maltogenic amylase, that is, suggesting an exo-mechanism for this enzyme.

PLANT PHYSIOLOGY, 1990

Sucrose esters and duvatrienediol diterpenes are principal constituents formed in and secreted ou... more Sucrose esters and duvatrienediol diterpenes are principal constituents formed in and secreted outside head cells of trichomes occurring on surfaces of Nicotiana tabacum. Using trichome-bearing epidermal peels prepared from midveins of N. tabacum cv T.l. 1068 leaves, we found that chlorsulfuron reduced and modified radiolabeling of sucrose ester acyl acids derived from branched-chain amino acid metabolism. The herbicide did not effect formation and exudation of diterpenes which are products of isoprenoid metabolism. Treatment with 1.0 micromolar chlorsulfuron affected 8.5-and 6.3-fold reductions in radiolabeling of methylvaleryl and methylbutyryl groups of sucrose esters, respectively, and concomitant increases of 9-and 9.8-fold in radiolabeling of straight chain valeryl and butyryl groups, respectively. These results and others indicate that inhibition of acetolactate synthase causes an accumulation of 2-oxo-butyric acid that is utilized by enzymes common to Leu biosynthesis to form 2-oxo-valeric acid. Coenzyme A (CoA) activation of this keto acid gives rise to butyryl CoA, which is utilized to form butyryl containing sucrose esters. Alternatively, reutilization of 2-oxo-valeric acid by the same enzymes followed by CoA activation leads to valeryl containing sucrose esters. We propose that in trichome secretory cells synthase, isomerase and dehydrogenase enzymes which catalyze Leu synthesis/degredation in most tissues, convert iso-branched, anteiso-branched and straight-chain keto acids in the formation of sucrose ester acyl groups. as previously described (9). CS, analytical grade (E.I. duPont 906 www.plantphysiol.org on August 10, 2018 -Published by Downloaded from

Phytochemistry, 1998

AbstractÐCompounds volatilized from plant tissues play important roles in plant-insect and plant-... more AbstractÐCompounds volatilized from plant tissues play important roles in plant-insect and plant-herbivore interactions and are important to food quality/preference, and to the perfume and¯avorant industries. While the chemistry of plant volatiles is well understood, less is known about the biosynthesis of this diverse group of compounds. This is particularly the case for non-terpenoid components such as volatile acyclic alcohols and their esters. Here we have studied metabolic pathways leading to formation of the anteiso-branched alcohol 4-methyl-1-hexanol volatilized by petal tissue of Nicotiana sylvestris. Evidence presented supports the involvement of steps in the pathways of both biosynthesis and degradation of isoleucine to form 2-oxo-3-methylvaleric acid then 2-methylbutyryl CoA. Results indicate that 2-methylbutyryl CoA is then elongated by addition of one acetate molecule via fatty acid synthase, followed by reduction to yield 4-methyl-1-hexanol. This pathway is in contrast to elongation of 2-oxo-3-methylvaleric acid via aketo acid elongation leading to the formation of 4-methylhexanoyl acyl groups of tobacco leaf-trichome-secreted sugar esters. #

Journal of Molecular Catalysis B: Enzymatic, 2011

The transglycosylation activity of barley α-amylase 1 (AMY1) and active site AMY1 subsite mutant ... more The transglycosylation activity of barley α-amylase 1 (AMY1) and active site AMY1 subsite mutant enzymes was investigated. We report here the transferase ability of the V47A, V47F, V47D and S48Y single mutants and V47K/S48G and V47G/S48D double mutant AMY1 enzymes in which the replaced amino acids play important role in substrate binding at subsites at −3 through −5. Although mutation

Journal of Inclusion Phenomena, 1984

Carbohydrate Research, 2012

DispersinB (DspB), a member of b-1,6-N-acetylglucosaminidase group of GH 20 glycoside hydrolases,... more DispersinB (DspB), a member of b-1,6-N-acetylglucosaminidase group of GH 20 glycoside hydrolases, catalyses the biofilm degradation of several human pathogenic microorganisms. DspB is a (b/a) 8 barrel protein, showing retaining cleavage mechanism towards oligomer and polymer substrates. A chromophore containing oligomer substrate series was used to study the DspB's mode of action. The hydrolysis reaction of b(1,6)-linked N-acetylglucosamine thiophenyl glycosides with degree of polymerisation of 2, 3, 4 and 5 was followed by reversed phase HPLC and progress curves were determined and analysed. Based on the analysis of process curves obtained from prolonged hydrolysis we assumed the presence of more productive binding modes resulting in parallel reactions followed by consecutive reaction steps. Strictly nonreducing-end specificity was observed, the presence of monomer, dimer and trimer nonreducing-end products was verified by MALDI-TOF MS. Another cleavage was suggested after the first glycosidic attack in the case of trimer, while two and three consecutive steps were possible in tetramer and pentamer hydrolyses, respectively. Chain lengthening increased catalytic efficiency (2.1?8.6 M À1 s À1 ) and calculated kinetic constants showed a similarly increasing tendency (1.0?6.7 Â 10 À3 min À1 ).

Carbohydrate Research, 1997

One-pot acetylation and subsequent partial acetolysis of (Y-, p-and y-cyclodextrins resulted in c... more One-pot acetylation and subsequent partial acetolysis of (Y-, p-and y-cyclodextrins resulted in crystalline peracetylated malto-hexaose, -heptaose, and -octaose, respectively. Prolonged acetolysis of P-cyclodextrin gave a mixture of acetylated maltooligosaccharides, from which peracetylated malto-triose, -tetraose, and -pentaose were isolated. The acetylated oligosaccharides were converted into cw-acetobromo derivatives, and then transformed into 4-nitrophenyl and 2-chloro-4-nitrophenyl P-glycosides. From the 4-nitrophenyl glycosides 4,60benzylidene derivatives were prepared, which were used together with the free glycosides as substrates of porcine pancreatic a-amylase. 0 1997 Elsevier Science Ltd. Kepwds: Acetolysis; Cyclodextrins; Maltooligosaccharide derivatives; cu-Amylase substrate * Corresponding author. ' Dedicated to Professor Dr. Hans Paulsen on the occasion of his 75th birthday.

Carbohydrate Research, 2011

Dispersin B (DspB) from Aggregatibacter actinomycetemcomitans is a β-hexosaminidase exhibiting bi... more Dispersin B (DspB) from Aggregatibacter actinomycetemcomitans is a β-hexosaminidase exhibiting biofilm detachment activity. A series of β-(1→6)-linked N-acetyl-D-glucosamine thiophenyl glycosides with degree of polymerisation (DP) of 2, 3, 4 and 5 were synthesized, and substrate specificity of DspB was studied on the obtained oligosaccharides. For oligomer synthesis a 1+2, 2+2, 1+4 coupling strategy was applied, using bromo-sugars as glycosyl donors. The formation of 1,2-trans interglycosidic bond has been ensured by 2-phtalimido protecting group; chloroacetyl group was installed to mask temporarily the 6-hydroxyl and acetate esters were applied as permanent protecting groups. Enzymatic studies revealed that DP of the GlcNAc oligomers strongly affected the hydrolysis rate, and the hydrolytic activity of DspB on the tetramer and pentamer have been found to be approximately 10-fold higher than that of the dimer. This fact indicates that four units are required for a strong binding at the active centre of DspB. The role of aromatic amino acids W237, Y187 and Y278 in substrate specificity and catalysis was also examined using mutant enzymes.

Biologia, 2008

Human salivary α-amylase (HSAmy) has three distinct functions relevant to oral health: (i) hydrol... more Human salivary α-amylase (HSAmy) has three distinct functions relevant to oral health: (i) hydrolysis of starch; (ii) binding to hydroxyapatite; and (iii) binding to bacteria (e.g. viridans streptococci). Oral bacteria utilize the starch hydrolyzing activity of HSAmy to derive their nutrients from dietary starch. Localized acid production by bacteria, through the metabolism of maltose generated by HSAmy, can lead to the dissolution of tooth enamel, a critical step in dental caries formation. HSAmy is a component of the acquired enamel pellicle and is used by Streptococcus gordonii to colonize the oral cavity. Although the active site of HSAmy for starch hydrolysis is well characterized, the regions responsible for the bacterial binding are yet to be defined. Since HSAmy possesses several secondary saccharide-binding sites in which aromatic residues are prominently located, we hypothesized that one of the secondary saccharide-binding sites harboring the aromatic residues W316 and W38...

Biochemistry, 2009

Some polysaccharide processing enzymes possess secondary carbohydrate binding sites situated on t... more Some polysaccharide processing enzymes possess secondary carbohydrate binding sites situated on the surface far from the active site. In barley alpha-amylase 1 (AMY1), two such sites, SBS1 and SBS2, are found on the catalytic (beta/alpha)(8)-barrel and the noncatalytic C-terminal domain, respectively. Site-directed mutagenesis of Trp(278) and Trp(279), stacking onto adjacent ligand glucosyl residues at SBS1, and of Tyr(380) and His(395), making numerous ligand contacts at SBS2, suggested that SBS1 and SBS2 act synergistically in degradation of starch granules. While SBS1 makes the major contribution to binding and hydrolysis of starch granules, SBS2 exhibits a higher affinity for the starch mimic beta-cyclodextrin. Compared to that of wild-type AMY1, the K(d) of starch granule binding by the SBS1 W278A, W279A, and W278A/W279A mutants thus increased 15-35 times; furthermore, the k(cat)/K(m) of W278A/W279A was 2%, whereas both affinity and activity for Y380A at SBS2 were 10% of the wild-type values. Dual site double and triple SBS1/SBS2 substitutions eliminated binding to starch granules, and the k(cat)/K(m) of W278A/W279A/Y380A AMY1 was only 0.4% of the wild-type value. Surface plasmon resonance analysis of mutants showed that beta-cyclodextrin binds to SBS2 and SBS1 with K(d,1) and K(d,2) values of 0.07 and 1.40 mM, respectively. A model that accounts for the observed synergy in starch hydrolysis, where SBS1 and SBS2 bind ordered and free alpha-glucan chains, respectively, thus targeting the enzyme to single alpha-glucan chains accessible for hydrolysis, is proposed. SBS1 and SBS2 also influence the kinetics of hydrolysis for amylose and maltooligosaccharides, the degree of multiple attack on amylose, and subsite binding energies.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2013

β-Amylase (EC 3.2.1.2), one of the main protein of the sweet potato, is an exo-working enzyme cat... more β-Amylase (EC 3.2.1.2), one of the main protein of the sweet potato, is an exo-working enzyme catalyzing the hydrolysis of α(1,4) glycosidic linkages in polysaccharides and removes successively maltose units from the non-reducing ends. The enzyme belongs to glycoside hydrolase GH14 family and inverts the anomeric configuration of the hydrolysis product. Multiple attack or processivity is an important property of polymer active enzymes and there is still limited information about the processivity of carbohydrate active enzymes. Action pattern and kinetic measurements of sweet potato β-amylase were made on a series of aromatic chromophor group-containing substrates (degree of polymerization DP 3-13) using HPLC method. Measured catalytic efficiencies increased with increasing DP of the substrates. Processive cleavage was observed on all substrates except the shortest pentamer. The mean number of steps without dissociation of enzyme-product complex increases with DP of substrate and reached 3.3 in case of CNPG11 indicating that processivity on longer substrates was more significant. A unique transglycosylation was observed on those substrates, which suffer processive cleavage and the substrates were re-built by the enzyme. Our results are the first presentation of a transglycosylation during an inverting glycosidase catalyzed hydrolysis. The yield of transglycosylation was remarkable high as shown in the change of the CNPG11 quantity. The CNPG11 concentration was doubled (from 0.24 to 0.54mM) in the early phase of the reaction.

Archives of Biochemistry and Biophysics, 1988

ABSTRACT A kutatás fő célkitűzése volt szénhidrátok és származékaik (védőcsoportokat tartalmazó v... more ABSTRACT A kutatás fő célkitűzése volt szénhidrátok és származékaik (védőcsoportokat tartalmazó vagy fehérjéhez kötött oligoszacharidok) MALDI-TOF tömegspektrometriás meghatározásához optimális kísérleti körülmények megállapítása és így a szénhidrátkémiai kutatások segítése. Ciklodextrin modellvegyületek alkalmazásával vizsgáltuk a minta előkészítés és felvitel valamint a kísérleti paraméterek változtatásának hatását a spektrumra. Tanulmányozzuk a mátrix anyagának, a minta koncentrációjának, a mintafelvitel módjának és a reflektron detektor érzékenység hatását a spektrum minőségére. Védőcsoportokat tartalmazó oligoszacharidok mérésekor számos törvényszerűséget figyeltünk meg a védőcsoportok stabilitása, a különböző mátrixok és kísérleti paraméterek között. A MALDI mérések segítették a termékek és melléktermékek azonosítását kémiai és enzimes oligoszacharid szintézisek esetében. Fragmentációs vizsgálatokkal kémiai és enzimatikus szintézissel előállított oligoszacharidok, valamint természetes eredetű amiláz inhibítorok szerkezet felderítését végeztük el. Meghatároztuk különböző neoglikoproteinek oligoszacharid tartalmát, amely meghatározó a sikeres immunválasz kiváltásában. Méréseinkkel támogattuk a különböző neoglikoprotein előállítási módszerek szisztematikus vizsgálatát. | The object of this research program was the application of MALDI-TOF MS in the field of carbohydrates and glycoconjugates. Cyclodextrins as model compounds were used to investigate the effect of the sample preparation methods and experimental parameters on the spectrum. The studied experimental parameters were: different matrix compounds, detector sensitivity, sample concentration and sample preparation methods. The stability of the protecting groups under the conditions of the evaporation/ionization was studied. The MALDI measurements assisted to identify products and byproducts of different chemical and enzymatic oligosaccharide syntheses (eg. sugar-sulfonates, sulfonic acid esters, PNP-glycosides and acarbose derivatives). Structural analysis and verification were made using post source decay fragmentation method in case of a tetrasaccharid component of N-glycanes, acarbose and its derivatives obtained by enzymatic synthesis and some natural alpha-amylase inhibitors. In addition we used this method for determination of molecular masses of neoglycoproteins.