Konstantin A. Shabalin

Enzymatic synthesis of β-xylanase substrates: transglycosylation reactions of the β-xylosidase from Aspergillus sp.

A beta-D-xylosidase with molecular mass of 250+/-5 kDa consisting of two identical subunits was purified to homogeneity from a cultural filtrate of Aspergillus sp. The enzyme manifested high transglycosylation activity in transxylosylation with p-nitrophenyl beta-D-xylopyranoside (PNP-X) as substrate, resulting in regio- and stereoselective synthesis of p-nitrophenyl (PNP) beta-(1-->4)-D-xylooligosaccharides with dp 2-7. All transfer products were isolated from the reaction mixtures by HPLC and their structures established by electrospray mass spectrometry and 1H and 13C NMR spectroscopy. The glycosides synthesised, beta-Xyl-1-->(4-beta-Xyl-1-->)(n)4-beta-Xyl-OC6H4NO2-p (n=1-5), were tested as chromogenic substrates for family 10 beta-xylanase from Aspergillus orizae (XynA) and family 11 beta-xylanase I from Trichoderma reesei (XynT) by reversed-phase HPLC and UV-spectroscopy techniques. The action pattern of XynA against the foregoing PNP beta-(1-->4)-D-xylooligosaccharides differed from that of XynT in that the latter released PNP mainly from short PNP xylosides (dp 2-3) while the former liberated PNP from the entire set of substrates synthesised.

Enzymatic properties of α-galactosidase from Trichoderma reesei in the hydrolysis of galactooligosaccharides

Enzymatic properties of the α-galactosidase (α-galactoside galactohydrolase, EC 3.2.1.22) from Trichoderma reesei in the hydrolysis of natural galactooligosaccharides and α-O-methyl D-galactopyranoside have been investigated in a wide range of substrate concentrations. The hydrolyses of α-O-methyl D-galactopyranoside and melibiose were inhibited by substrate at concentrations higher than 100 mM while in the hydrolysis of raffinose and stachyose such an effect was not observed. It was shown by 1H and 13C NMR spectroscopy and HPLC techniques that inhibition by the excess of α-O-methyl D-galactopyranoside or melibiose strongly correlated with formation of transglycosylation products. The product of autocondensation reaction with α-O-methyl D-galactopyranoside as substrate was found to be α-O-methyl galactopyranosyl-1,6-D-galactopyranoside. The stereochemical course of stachyose hydrolysis has been determined. The enzyme catalyses the hydrolysis with retention of anomeric configuration and is assumed to operate via a double displacement mechanism. Simultaneous hydrolysis of stachyose and raffinose effected by the α-D-galactosidase was studied by direct 1H NMR measurements. Cleavage of the terminal galactose residue of stachyose was found to be the rate-limiting step. Formation constants of enzyme-substrate complex for stachyose and raffinose were calculated. The suggested model can be used for simulating the two-substrate system and predicting the extent of stachyose hydrolysis.

Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells.

Background: Nicotinamide riboside (NR) and nicotinic acid riboside (NAR) can serve as precursors of NAD in human cells. Results: Human cells generate and release NR and NAR. Conclusion: NR and NAR are authentic intermediates of human NAD metabolism. Significance: Different cell populations might support each others NAD pools by providing ribosides as NAD precursors. NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5′-nucleotidases (5′-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5′-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5′-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each others NAD supply by providing alternative precursors.

An α-L-fucosidase from Thermus sp. with unusually broad specificity

An α-L-fucosidase (E.C. 3.2.1.51) exhibiting a wide aglycon specificity expressed in ability of cleaving α1 → 6-, α1 →3-, α1 → 4-, and α1 → 2-O-fucosyl bonds in fucosylated oligosaccharides, has been isolated from culture filtrate of Thermus sp. strain Y5. The α-L-fucosidase hydrolyzes p-nitrophenyl α-L-fucopyranoside with Vmax of 12.0 ± 0.1 μM/min/mg and Km = 0.20 ± 0.05 mM and is able to cleave off about 90% of total L-fucose from pronase-treated fractions of fucosyl-containing glycoproteins and about 30% from the native glycoproteins. The purified enzyme is a tetramer with a molecular mass of 240 ± 10 kDa consisting of four identical subunits with a molecular mass of 61.0 ± 0.5 kDa. The N-terminal sequence showed homology to some α-L-fucosidases from microbial and plant sources. Hydrolysis of p-nitrophenyl α-L-fucopyranoside occurs with retention of the anomeric configuration. Transglycosylating activity of the α-L-fucosidase was demonstrated in reactions with such acceptors as alcohols, N-acetylglucosamine and N-acetylgalactosamine while no transglycosylation products were observed in the reaction with p-nitrophenyl α-L-fucopyranoside. The enzyme can be classified in glycosyl hydrolase family 29.

Transglycosylating and hydrolytic activities of the β-mannosidase from Trichoderma reesei

A purified beta-mannosidase (EC 3.2.1.25) from the fungus Trichoderma reesei has been identified as a member of glycoside hydrolase family 2 through mass spectrometry analysis of tryptic peptides. In addition to hydrolysis, the enzyme catalyzes substrate transglycosylation with p-nitrophenyl beta-mannopyranoside. Structures of the major and minor products of this reaction were identified by NMR analysis as p-nitrophenyl mannobiosides and p-nitrophenyl mannotriosides containing beta-(1-->4) and beta-(1-->3) linkages. The rate of donor substrate hydrolysis increased in presence of acetonitrile and dimethylformamide, while transglycosylation was weakly suppressed by these organic solvents. Differential ultraviolet spectra of the protein indicate that a rearrangement of the hydrophobic environment of the active site following the addition of the organic solvents may be responsible for this hydrolytic activation.

Biochemical characterization of Aspergillus awamori exoinulinase: substrate binding characteristics and regioselectivity of hydrolysis

1H-NMR analysis was applied to investigate the hydrolytic activity of Aspergillus awamori inulinase. The obtained NMR signals and deduced metabolite pattern revealed that the enzyme cleaves off only fructose from inulin and does not possess transglycosylating activity. Kinetics for the enzyme hydrolysis of inulooligosaccharides with different degree of polymerization (d.p.) were recorded. The enzyme hydrolyzed both beta2,1- as well as beta2,6-fructosyl linkages in fructooligosaccharides. From the k(cat)/K(m) ratios obtained with inulooligosaccharides with d.p. from 2 to 7, we deduce that the catalytic site of the inulinase contains at least five fructosyl-binding sites and can be classified as exo-acting enzyme. Product analysis of inulopentaose and inulohexaose hydrolysis by the Aspergillus inulinase provided no evidence for a possible multiple-attack mode of action, suggesting that the enzyme acts exclusively as an exoinulinase.

Enzymatic activity and β-galactomannan binding property of β-mannosidase from Trichoderm reesei

An extracellular 105-kDa β-mannosidase (β-d-mannoside-mannohydrolase, E.C. 3.2.1.25) was purified to homogeneity from culture filtrate of Trichoderma reesei. Specific activity of the β-mannosidase toward p-nitrophenyl-β-d-mannopyranoside was 3.2 U/mg at the optimal pH 3.5 (Km = 0.12 mM, kcat = 2.95 × 10−3 μmol min/μg. An additional β-galactomannan (GM) binding site of the enzyme was found on the basis of kinetic studies. The enzyme GM dissociation constant (KD) was 1.21 mg/ml. β-1,4-mannooligosaccharides inhibited the binding of the enzyme to galactomannan. The inhibition constant of the sorption decreased with increasing of the β-1,4-mannooligosaccharide length. Mannose, the competitive inhibitor of the β-mannosidase in hydrolysis of p-nitrophenyl-β-d-mannopyranoside, did not inhibit sorption of the enzyme on β-GM. Chitin, xylan, raw starch, and microcrystalline cellulose had no affinity to the β-mannosidase. The enzyme hydrolyzed β-1,4-mannooligosaccharides with the rate depending on the chain length and liberated mannose from soluble and insoluble fractions of β-GM from locust beans with initial rates of 0.3 and 0.05 μmol min/ml U, respectively.

The carbohydrate moiety of α-galactosidase from Trichoderma reesei

Abstracta-Galactosidase from Trichoderma reesei is a glycoprotein that contains O- and N-linked carbohydrate chains. There are 6 O-linked glycans per protein molecule that are linked to serine and threonine and can be released by b-elimination. Among these are monomers: D-glucose, D-mannose, and D-galactose; dimers: a1-6 D-mannopyranosyl- a-D-glycopyranoside and a1-6 D-glucopyranosyl- a-D-galactopyranoside and one trimer: a-D-glucopyranosyl- a1-2 D-mannopyranosyl- a1-6 D-galac-topyranoside. N-linked glycans are of the mannose-rich type and may be released by treating the protein with Endo- b-N-acetyl glycosaminidase F or by hydrozinolysis. The enzyme was deglycosylated with Endo- b- N-acetyl glycosaminidase F as well as with a number of exoglycosidases that partially remove the terminal residues of O-linked glycans. The effect of enzymatic deglycosylation on the properties of a-galactosidase has been considered. The effects of tunicamycin and 2-deoxyglucose on the secretion and glycosylation of the enzyme during culture growth have been analysed. The presence of two glycoforms of a-glactosidase differing in the number of N-linked carbohydrate chains and the microheterogeneity of the carbohydrate moiety of the enzyme are described.

Enzymatic synthesis of 4-methylumbelliferyl (1→3)-β-d-glucooligosaccharides—new substrates for β-1,3-1,4-d-glucanase

Abstract The transglycosylation reactions catalyzed by β-1,3- d -glucanases (laminaranases) were used to synthesize a number of 4-methylumbelliferyl (MeUmb) (1→3)-β- d -gluco-oligosaccharides having the common structure [β- d -Glcp-(1→3)]n-β- d -Glcp-MeUmb, where n=1–5. The β-1,3- d- glucanases used were purified from the culture liquid of Oerskovia sp. and from a homogenate of the marine mollusc Spisula sachalinensis. Laminaran and curdlan were used as (1→3)-β- d -glucan donor substrates, while MeUmb-β- d -glucoside (MeUmbGlcp) was employed as a transglycosylation acceptor. Modification of [β- d -Glcp-(1→3)]2-β- d -Glcp-MeUmb (MeUmbG3) gives 4,6-O-benzylidene- d -glucopyranosyl or 4,6-O-ethylidene- d -glucopyranosyl groups at the non-reducing end of artificial oligosaccharides. The structures of all oligosaccharides obtained were solved by 1H and 13C NMR spectroscopy and electrospray tandem mass spectrometry. The synthetic oligosaccharides were shown to be substrates for a β-1,3-1,4- d -glucanase from Rhodothermus marinus, which releases MeUmb from β-di- and β-triglucosides and from acetal-protected β-triglucosides. When acting upon substrates with d.p.>3, the enzyme exhibits an endolytic activity, primarily cleaving off MeUmbGlcp and MeUmbG2.

1-O-Acetyl-β-d-galactopyranose: a novel substrate for the transglycosylation reaction catalyzed by the β-galactosidase from Penicillium sp.

Abstract 1- O -Acetyl-β- d -galactopyranose (AcGal), a new substrate for β-galactosidase, was synthesized in a stereoselective manner by the trichloroacetimidate procedure. Kinetic parameters ( K M and k cat ) for the hydrolysis of 1- O -acetyl-β- d -galactopyranose catalyzed by the β- d -galactosidase from Penicillium sp. were compared with similar characteristics for a number of natural and synthetic substrates. The value for k cat in the hydrolysis of AcGal was three orders of magnitude greater than for other known substrates. The β-galactosidase hydrolyzes AcGal with retention of anomeric configuration. The transglycosylation activity of the β- d -galactosidase in the reaction of AcGal and methyl β- d -galactopyranoside ( 1 ) as substrates was investigated by 1 H NMR spectroscopy and HPLC techniques. The transglycosylation product using AcGal as a substrate was β- d -galactopyranosyl-(1→6)-1- O -acetyl-β- d -galactopyranose (with a yield of ∼70%). In the case of 1 as a substrate, the main transglycosylation product was methyl β- d -galactopyranosyl-(1→6)-β- d -galactopyranoside. Methyl β- d -galactopyranosyl-(1→3)-β- d -galactopyranoside was found to be minor product in the latter reaction.