Yuji Honda

The First Glycosynthase Derived from an Inverting Glycoside Hydrolase

Reducing end xylose-releasing exooligoxylanase (Rex, EC 3.2.1.156) is an inverting GH that hydrolyzes xylooligosaccharides (≥X3) to release X1 at their reducing end. The wild-type enzyme exhibited the Hehre resynthesis hydrolysis mechanism, in which α-X2F was hydrolyzed to X2 and HF in the presence of X1 as an acceptor molecule. However, the transglycosidation product (X3) was not detectable in the reaction. To convert reducing end xylose-releasing exooligoxylanase to glycosynthase, derivatives with mutations in the catalytic base (Asp-263) were constructed by saturation random mutagenesis. Nine amino acid residue mutants (Asp-263 to Gly, Ala, Val, Thr, Leu, Asn, Cys, Pro, or Ser) were found to possess glycosynthase activity forming X3 from α-X2F and X1. Among them, D263C showed the highest level of X3 production, and D263N exhibited the fastest consumption of α-X2F. The D263C mutant showed 10-fold lower hydrolytic activity than D263N, resulting in the highest yield of X3. X2 was formed from the early stage of the reaction of the D263C mutant, indicating that a portion of the X3 formed by condensation was hydrolyzed before its release from the enzyme. To acquire glycosynthase activity from inverting enzymes, it is important to minimize the decrease in F–-releasing activity while maximizing the decrease in the hydrolytic activity. The present study expands the possibility of conversion of glycosynthases from inverting enzymes.

1,2‐α‐l‐Fucosynthase: A glycosynthase derived from an inverting α‐glycosidase with an unusual reaction mechanism

Fucosyloligosaccharides have great therapeutic potential. Here we present a new route for synthesizing a Fucα1,2Gal linkage by introducing glycosynthase technology into 1,2‐α‐l‐fucosidase. The enzyme adopts a unique reaction mechanism, in which asparagine‐423 activated by aspartic acid‐766 acts as a base while asparagine‐421 fixes both a catalytic water and glutamic acid‐566 (an acid) in the proper orientations. Glycosynthase activity of N421G, N423G, and D766G mutants was examined using β‐fucosyl fluoride and lactose, and among them, the D766G mutant most effectively synthesized 2′‐fucosyllactose. 1,2‐α‐l‐Fucosynthase is the first glycosynthase derived from an inverting α‐glycosidase and from a glycosidase with an unusual reaction mechanism.

A family 8 glycoside hydrolase from Bacillus halodurans C-125 (BH2105) is a reducing end xylose-releasing exo-oligoxylanase

The gene encoding family 8 glycoside hydrolases from Bacillus halodurans C-125 (BH2105), an alkalophilic bacterium with a known genomic sequence, was expressed in Escherichia coli. The protein was expressed with the intact N-terminal sequence, suggesting that it did not possess a signal peptide and that it was an intracellular enzyme. The recombinant enzyme showed no hydrolytic activity on xylan, whereas it had been annotated as xylanase Y. It hydrolyzed xylooligosaccharide whose degree of polymerization is greater than or equal to 3 in an exo-splitting manner with anomeric inversion, releasing the xylose unit at the reducing end. Judging from its substrate specificity and reaction mechanism, we named the enzyme reducing end xylose-releasing exo-oligoxylanase (Rex). Rex was found to utilize only the β-anomer of the substrate to form β-xylose and α-xylooligosaccharide. The optimum pH of the enzymatic reaction (6.2–7.3) was found in the neutral range, a range beneficial for intracellular enzymes. The genomic sequence suggests that B. halodurans secretes two endoxylanases and possesses two α-arabinofuranosidases, one α-glucuronidase, and three β-xylosidases intracellularly in addition to Rex. The extracellular enzymes supposedly hydrolyze xylan into arabino/glucurono-xylooligosaccharides that are then transported into the cells. Rex may play a role as a key enzyme in intracellular xylan metabolism in B. halodurans by cleaving xylooligosaccharides that were produced by the action of other intracellular enzymes from the arabino/glucurono-xylooligosaccharides.

Substrate binding subsites of chitinase from barley seeds and lysozyme from goose egg white

Substrate binding subsites of barley chitinase and goose egg white lysozyme were comparatively investigated by kinetic analysis using N-acetylglucosamine oligosaccharide as the substrate. The enzymatic hydrolysis of hexasaccharide was monitored by HPLC, and the reaction time-course was analyzed by the mathematical model, in which six binding subsites (B, C, D, E, F, and G) and bond cleavage between sites D and E are postulated. In this model, all of the possible binding modes of substrate and products are taken into consideration assuming a rapid equilibrium in the oligosaccharide binding processes. To estimate the binding free energy changes of the subsites, time-course calculation was repeated with changing the free energy values of individual subsites, until the calculated time-course was sufficiently fitted to the experimental one. The binding free energy changes of the six binding subsites, B, C, D, E, F and G, which could give a calculated time-course best fitted to the experimental, were 0.0, -5.0, +4.1, -0.5, -3.8, and -2.0 kcal/mol for barley chitinase, and -0.5, -2.2, +4.2, -1.5, -2.6, and -2.8 kcal/mol for goose egg white lysozyme. The binding mode predicted from the p-nitrophenyl-penta-N-acetylchitopentaoside splitting pattern for each enzyme was also analyzed by the identical subsite model. Using the free energy values listed above, the binding mode distribution calculated was fitted to the experimental with a slight modification of free energy value at site G. We concluded that the binding subsite model described above reflects the substantial mechanism of substrate binding for both enzymes. The relatively large disparity in free energy value at site C between these enzymes may be due to the different secondary structures of polypeptide segments interacting with the sugar residue at site C.

Alternative strategy for converting an inverting glycoside hydrolase into a glycosynthase

The tyrosine residue Y198 is known to support a nucleophilic water molecule with the general base residue, D263, in the reducing-end xylose-releasing exo-oligoxylanase (Rex). A mutation in the tyrosine residue changing it into phenylalanine caused a drastic decrease in the hydrolytic activity and a small increase in the F(-) releasing activity from alpha-xylobiosyl fluoride in the presence of xylose. In contrast, mutations at D263 resulted in the decreased F(-) releasing activity. As a result of the high F(-) releasing activity and low hydrolytic activity, Y198F of Rex accumulates a large amount of product during the glycosynthase reaction. We propose a novel method for producing a glycosynthase from an inverting glycoside hydrolase by mutating a residue that holds the nucleophilic water molecule with the general base residue while keeping the general base residue intact.

Structural Basis for the Specificity of the Reducing End Xylose-releasing Exo-oligoxylanase from Bacillus halodurans C-125

Reducing end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125 (Rex) hydrolyzes xylooligosaccharides whose degree of polymerization is greater than or equal to 3, releasing the xylose unit at the reducing end. It is a unique exo-type glycoside hydrolase that recognizes the xylose unit at the reducing end in a very strict manner, even discriminating the β-anomeric hydroxyl configuration from the α-anomer or 1-deoxyxylose. We have determined the crystal structures of Rex in unliganded and complex forms at 1.35–2.20-Å resolution and revealed the structural aspects of its three subsites ranging from –2 to +1. The structure of Rex was compared with those of endo-type enzymes in glycoside hydrolase subfamily 8a (GH-8a). The catalytic machinery of Rex is basically conserved with other GH-8a enzymes. However, subsite +2 is blocked by a barrier formed by a kink in the loop before helix α10. His-319 in this loop forms a direct hydrogen bond with the β-hydroxyl of xylose at subsite +1, contributing to the specific recognition of anomers at the reducing end.

1,3-1,4-α-l-Fucosynthase That Specifically Introduces Lewis a/x Antigens into Type-1/2 Chains

Background: Regiospecific installation of α-l-fucosyl residue into glycoconjugates is quite difficult. Results: A glycosynthase mutant of 1,3-1,4-α-l-fucosidase specifically synthesized Lewis a/x trisaccharides using Galβ1–3/4GlcNAc as acceptors. Conclusion: Structural studies provide a rationale to explain the unusually strict substrate specificity exhibited by the enzyme. Significance: A new enzymatic route for specifically introducing Lewis a/x epitopes into type-1/2 chains becomes available. α-l-Fucosyl residues attached at the non-reducing ends of glycoconjugates constitute histo-blood group antigens Lewis (Le) and ABO and play fundamental roles in various biological processes. Therefore, establishing a method for synthesizing the antigens is important for functional glycomics studies. However, regiospecific synthesis of glycosyl linkages, especially α-l-fucosyl linkages, is quite difficult to control both by chemists and enzymologists. Here, we generated an α-l-fucosynthase that specifically introduces Lea and Lex antigens into the type-1 and type-2 chains, respectively; i.e. the enzyme specifically accepts the disaccharide structures (Galβ1–3/4GlcNAc) at the non-reducing ends and attaches a Fuc residue via an α-(1,4/3)-linkage to the GlcNAc. X-ray crystallographic studies revealed the structural basis of this strict regio- and acceptor specificity, which includes the induced fit movement of the catalytically important residues, and the difference between the active site structures of 1,3-1,4-α-l-fucosidase (EC 3.2.1.111) and α-l-fucosidase (EC 3.2.1.51) in glycoside hydrolase family 29. The glycosynthase developed in this study should serve as a potentially powerful tool to specifically introduce the Lea/x epitopes onto labile glycoconjugates including glycoproteins. Mining glycosidases with strict specificity may represent the most efficient route to the specific synthesis of glycosidic bonds.

Reaction mechanism of chitobiose phosphorylase from Vibrio proteolyticus : Identification of family 36 glycosyltransferase in Vibrio

A family 36 glycosyltransferase gene was cloned from Vibrio proteolyticus. The deduced amino acid sequence showed a high degree of identity with ChBP (chitobiose phosphorylase) from another species, Vibrio furnissii. The recombinant enzyme catalysed the reversible phosphorolysis of (GlcNAc)2 (chitobiose) to form 2-acetamide-2-deoxy-alpha-D-glucose 1-phosphate [GlcNAc-1-P] and GlcNAc, but showed no activity on cellobiose, indicating that the enzyme was ChBP, not cellobiose phosphorylase. In the synthetic reaction, the ChBP was active with alpha-D-glucose 1-phosphate as the donor substrate as well as GlcNAc-1-P to produce beta-D-glucosyl-(1-->4)-2-acetamide-2-deoxy-D-glucose with GlcNAc as the acceptor substrate. The enzyme allowed aryl-beta-glycosides of GlcNAc as the acceptor substrate with 10-20% activities of GlcNAc. Kinetic parameters of (GlcNAc)2 in the phosphorolysis and GlcNAc-1-P in the synthetic reaction were determined as follows: phosphorolysis, k(0)=5.5 s(-1), K(m)=2.0 mM; synthetic reaction, k(0)=10 s(-1), K(m)=14 mM, respectively. The mechanism of the phosphorolytic reaction followed a sequential Bi Bi mechanism, as frequently observed with cellobiose phosphorylases. Substrate inhibition by GlcNAc was observed in the synthetic reaction. The enzyme was considered a unique biocatalyst for glycosidation.

Lacto-N-biosidase encoded by a novel gene of Bifidobacterium longum subspecies longum shows unique substrate specificity and requires a designated chaperone for its active expression.

Background: Phenotypically lacto-N-biosidase-positive Bifidobacterium longum JCM1217 does not possess a gene homologous to previously identified lacto-N-biosidase. Results: Hypothetical proteins BLLJ_1505 and BLLJ_1506 encode lacto-N-biosidase and its designated chaperone, respectively. Conclusion: The enzyme showed unique and unexpected substrate specificity. Significance: The enzyme is important for understanding how B. longum consumes human milk oligosaccharides and also may serve as a new tool in glycobiology. Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N-tetraose (Galβ1–3GlcNAcβ1–3Galβ1–4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Galβ1–3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase+ strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAcβ1–3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc), and sialyllacto-N-tetraose a (Neu5Acα2–3Galβ1–3GlcNAcβ1–3Galβ1–4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-β-lacto-N-bioside I (Galβ1–3GlcNAcβ-pNP) and GalNAcβ1–3GlcNAcβ-pNP. GalNAcβ1–3GlcNAcβ linkage has been found in O-mannosyl glycans of α-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca2+ and Mg2+) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.

Kinetic evidence related to substrate-assisted catalysis of family 18 chitinases

The hydrolytic reaction of family 18 chitinase has been considered to occur via substrate assisted catalysis. To kinetically investigate the enzyme reaction mechanism, we synthesized compounds designed to reduce the polarization of the carbonyl in N‐acetyl group, GlcNAc‐GlcN(TFA)‐UMB (2) and GlcNAc‐GlcN(TAc)‐UMB (3). Kinetic parameters in the hydrolysis of these compounds by chitinase A from Serratia marcescens (ChiA) were compared with those from the hydrolysis of (GlcNAc)2‐UMB (1). The k cat of 2 was 3.4% of 1, but the K m of 2 was 10‐fold that of 1. In contrast, the k cat of 3 was only 0.3% of that of 1, and the two reactions had an identical K m. The drastic decreases in k cat were probably due to the weak nucleophilic activity of the C2‐N‐trifluoroacetamide and N‐thioacetamide groups at reducing ends of compounds 2 and 3, respectively. These results indicate that the anchimeric assistance of the C2 N‐acetamide group at GlcNAc plays a key role in the hydrolytic reactions catalyzed by family 18 chitinases.