Takayuki Ohnuma

Transglycosylation reaction catalyzed by a class V chitinase from cycad, Cycas revoluta: a study involving site-directed mutagenesis, HPLC, and real-time ESI-MS.

Class V chitinase from cycad, Cycas revoluta, (CrChi-A) is the first plant chitinase that has been found to possess transglycosylation activity. To identify the structural determinants that bring about transglycosylation activity, we mutated two aromatic residues, Phe166 and Trp197, which are likely located in the acceptor binding site, and the mutated enzymes (F166A, W197A) were characterized. When the time-courses of the enzymatic reaction toward chitin oligosaccharides were monitored by HPLC, the specific activity was decreased to about 5-10% of that of the wild type and the amounts of transglycosylation products were significantly reduced by the individual mutations. From comparison between the reaction time-courses obtained by HPLC and real-time ESI-MS, we found that the transglycosylation reaction takes place under the conditions used for HPLC but not under the ESI-MS conditions. The higher substrate concentration (5 mM) used for the HPLC determination is likely to bring about chitinase-catalyzed transglycosylation. Kinetic analysis of the time-courses obtained by HPLC indicated that the sugar residue affinity of +1 subsite was strongly reduced in both mutated enzymes, as compared with that of the wild type. The IC(50) value for the inhibitor allosamidin determined by real-time ESI-MS was not significantly affected by the individual mutations, indicating that the state of the allosamidin binding site (from -3 to -1 subsites) was not changed in the mutated enzymes. We concluded that the aromatic side chains of Phe166 and Trp197 in CrChi-A participate in the transglycosylation acceptor binding, thus controlling the transglycosylation activity of the enzyme.

Cloning and characterization of a small family 19 chitinase from moss (Bryum coronatum)

Chitinase-A (BcChi-A) was purified from a moss, Bryum coronatum, by several steps of column chromatography. The purified BcChi-A was found to be a molecular mass of 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 3.5. A cDNA encoding BcChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1012 nucleotides and encoded an open reading frame of 228 amino acid residues. The predicted mature BcChi-A consists of 205 amino acid residues and has a molecular weight of 22,654. Sequence analysis indicated that BcChi-A is glycoside hydrolase family-19 (GH19) chitinase lacking loops I, II, IV and V, and a C-terminal loop, which are present in the catalytic domain of plant class I and II chitinases. BcChi-A is a compact chitinase that has the fewest loop regions of the GH19 chitinases. Enzymatic experiments using chitooligosaccharides showed that BcChi-A has higher activity toward shorter substrates than class II enzymes. This characteristic is likely due to the loss of the loop regions that are located at the end of the substrate-binding cleft and would be involved in substrate binding of class II enzymes. This is the first report of a chitinase from mosses, nonvascular plants.

Chitin oligosaccharide binding to a family GH19 chitinase from the moss Bryum coronatum

Substrate binding of a family GH19 chitinase from a moss species, Bryum coronatum (BcChi‐A, 22 kDa), which is smaller than the 26 kDa family GH19 barley chitinase due to the lack of several loop regions (‘loopless’), was investigated by oligosaccharide digestion, thermal unfolding experiments and isothermal titration calorimetry (ITC). Chitin oligosaccharides [β‐1,4‐linked oligosaccharides of N‐acetylglucosamine with a polymerization degree of n, (GlcNAc)n, n = 3–6] were hydrolyzed by BcChi‐A at rates in the order (GlcNAc)6 > (GlcNAc)5 > (GlcNAc)4 >> (GlcNAc)3. From thermal unfolding experiments using the inactive BcChi‐A mutant (BcChi‐A‐E61A), in which the catalytic residue Glu61 is mutated to Ala, we found that the transition temperature (Tm) was elevated upon addition of (GlcNAc)n (n = 2–6) and that the elevation (ΔTm) was almost proportional to the degree of polymerization of (GlcNAc)n. ITC experiments provided the thermodynamic parameters for binding of (GlcNAc)n (n = 3–6) to BcChi‐A‐E61A, and revealed that the binding was driven by favorable enthalpy changes with unfavorable entropy changes. The change in heat capacity (ΔCp°) for (GlcNAc)6 binding was found to be relatively small (−105 ± 8 cal·K−1·mol−1). The binding free energy changes for (GlcNAc)6, (GlcNAc)5, (GlcNAc)4 and (GlcNAc)3 were determined to be −8.5, −7.9, −6.6 and −5.0 kcal·mol−1, respectively. Taken together, the substrate binding cleft of BcChi‐A consists of at least six subsites, in contrast to the four‐subsites binding cleft of the ‘loopless’ family 19 chitinase from Streptomyces coelicolor.

Crystal structure and chitin oligosaccharide-binding mode of a 'loopful' family GH19 chitinase from rye, Secale cereale, seeds

The substrate‐binding mode of a 26‐kDa GH19 chitinase from rye, Secale cereale, seeds (RSC‐c) was investigated by crystallography, site‐directed mutagenesis and NMR spectroscopy. The crystal structure of RSC‐c in a complex with an N‐acetylglucosamine tetramer, (GlcNAc)4, was successfully solved, and revealed the binding mode of the tetramer to be an aglycon‐binding site, subsites +1, +2, +3, and +4. These are the first crystallographic data showing the oligosaccharide‐binding mode of a family GH19 chitinase. From HPLC analysis of the enzymatic reaction products, mutation of Trp72 to alanine was found to affect the product distribution obtained from the substrate, p‐nitrophenyl penta‐N‐acetyl‐β‐chitopentaoside. Mutational experiments confirmed the crystallographic finding that the Trp72 side chain interacts with the +4 moiety of the bound substrate. To further confirm the crystallographic data, binding experiments were also conducted in solution using NMR spectroscopy. Several signals in the 1H–15N HSQC spectrum of the stable isotope‐labeled RSC‐c were affected upon addition of (GlcNAc)4. Signal assignments revealed that most signals responsive to the addition of (GlcNAc)4 are derived from amino acids located at the surface of the aglycon‐binding site. The binding mode deduced from NMR binding experiments in solution was consistent with that from the crystal structure.

A plant class V chitinase from a cycad (Cycas revoluta): Biochemical characterization, cDNA isolation, and posttranslational modification

Chitinase-A (CrChi-A) was purified from leaf rachises of Cycas revoluta by several steps of column chromatography. It was found to be a glycoprotein with a molecular mass of 40 kDa and an isoelectric point of 5.6. CrChi-A produced mainly (GlcNAc)(3) from the substrate (GlcNAc)(6) through a retaining mechanism. More interestingly, CrChi-A exhibited transglycosylation activity, which has not been observed in plant chitinases investigated so far. A cDNA encoding CrChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction procedures. It consisted of 1399 nucleotides and encoded an open reading frame of 387-amino-acid residues. Sequence analysis indicated that CrChi-A belongs to the group of plant class V chitinases. From peptide mapping and mass spectrometry of the native and recombinant enzyme, we found that an N-terminal signal peptide and a C-terminal extension were removed from the precursor (M1-A387) to produce a mature N-glycosylated protein (Q24-G370). This is the first report on a plant chitinase with transglycosylation activity and posttranslational modification of a plant class V chitinase.

Chitin-Related Enzymes in Agro-Biosciences

Plants utilized for agricultural productions interact with insects, fungi, and bacteria under the field conditions, affecting thereby their productivity. Since chitin and its derivatives play important roles in the interactions between these organisms, chitin-related enzymes are effective tools or drug targets for controlling the interactions. Thus, the molecular biology, protein chemistry, and enzymology of the chitin-related enzymes have been intensively studied by many investigators. Identifications and classifications of the genes encoding chitin synthetases, chitinases, chitosanases, and chitin deacetylases in these organisms were conducted, and their physiological functions were defined by knockdown, knockout, or overexpression of the corresponding genes. Recombinant enzyme productions and mutation studies are also being conducted to understand their structure and function. All of these studies have opened the way to efficiently utilize these enzyme tools for enhancing the agricultural productions.

Complete subsite mapping of a “loopful” GH19 chitinase from rye seeds based on its crystal structure

Crystallographic analysis of a mutated form of “loopful” GH19 chitinase from rye seeds a double mutant RSC‐c, in which Glu67 and Trp72 are mutated to glutamine and alanine, respectively, (RSC‐c‐E67Q/W72A) in complex with chitin tetrasaccharide (GlcNAc)4 revealed that the entire substrate‐binding cleft was completely occupied with the sugar residues of two (GlcNAc)4 molecules. One (GlcNAc)4 molecule bound to subsites −4 to −1, while the other bound to subsites +1 to +4. Comparisons of the main chain conformation between liganded RSC‐c‐E67Q/W72A and unliganded wild type RSC‐c suggested domain motion essential for catalysis. This is the first report on the complete subsite mapping of GH19 chitinase.

A flexible loop controlling the enzymatic activity and specificity in a glycosyl hydrolase family 19 endochitinase from barley seeds (Hordeum vulgare L.).

To examine the role of the loop structure consisting of residues 70-82 (70-82 loop) localized to +3/4 subsite of the substrate binding cleft of a family GH-19 endochitinase from barley seeds, Trp72 and Trp82 were mutated, and the mutated enzymes (W72A, W82A, and W72A/W82A) were characterized. Thermal stability and specific activities toward glycol chitin and chitin hexasaccharide were significantly affected by the individual mutations. When N-acetylglucosamine hexamer was hydrolyzed by the wild type, the beta-anomer of the substrate was preferentially hydrolyzed, producing the trimer predominantly and the dimer and tetramer in lesser amounts. When the mutated enzymes were used instead of the wild type, the enzyme cleavage sites in the hexamer substrate were clearly shifted, and the beta-anomer selectivity was eliminated. The mutation effects on the enzymatic activity and stability were much more substantial in W82A than in W72A, but surprisingly the effects of the W82A/W72A double mutation were intermediate between those of the two single mutations. A molecular dynamics simulation of the wild type and the Trp-mutated enzymes indicated that the 70-82 loop becomes more flexible upon mutation and the flexibility increases in the order of W72A, W72A/W82A and W82A. We conclude that Trp72 interacts with the sugar residue but Trp82 modulates the loop flexibility, which controls the protein stability and enzymatic properties. These tryptophan residues are likely to interact with each other, resulting in the non-additivity of mutational effects.

Introduction of a tryptophan side chain into subsite +1 enhances transglycosylation activity of a GH-18 chitinase from Arabidopsis thaliana, AtChiC

A tryptophan side chain was introduced into subsite +1 of family GH-18 (class V) chitinases from Nicotiana tabacum and Arabidopsis thaliana (NtChiV and AtChiC, respectively) by the mutation of a glycine residue to tryptophan (G74W-NtChiV and G75W-AtChiC). The specific activity toward glycol chitin of the two mutant enzymes was 70-71% of that of the wild type. Using chitin oligosaccharides, (GlcNAc)(n) (n = 4, 5 and 6), as the substrates, we found the transglycosylation reaction to be significantly enhanced in G74W-NtChiV and G75W-AtChiC when compared with the corresponding wild-type enzymes. The introduced tryptophan side chain might protect the oxazolinium ion intermediate from attack by a nucleophilic water molecule. The enhancement of transglycosylation activity was much more distinct in G75W-AtChiC than in G74W-NtChiV. Nuclear magnetic resonance titration experiments using the inactive double mutants, E115Q/G74W-NtChiV and E116Q/G75W-AtChiC revealed that the association constant of (GlcNAc)(5) was considerably larger for the latter. Amino acid substitutions at the acceptor binding site might have resulted in the larger association constant for G75W-AtChiC, giving rise to the higher transglycosylation activity of G75W-AtChiC.

A glycosynthase derived from an inverting GH19 chitinase from the moss Bryum coronatum

BcChi-A, a GH19 chitinase from the moss Bryum coronatum, is an endo-acting enzyme that hydrolyses the glycosidic bonds of chitin, (GlcNAc)(n) [a β-1,4-linked polysaccharide of GlcNAc (N-acetylglucosamine) with a polymerization degree of n], through an inverting mechanism. When the wild-type enzyme was incubated with α-(GlcNAc)2-F [α-(GlcNAc)(2) fluoride] in the absence or presence of (GlcNAc)(2), (GlcNAc)(2) and hydrogen fluoride were found to be produced through the Hehre resynthesis-hydrolysis mechanism. To convert BcChi-A into a glycosynthase, we employed the strategy reported by Honda et al. [(2006) J. Biol. Chem. 281, 1426-1431; (2008) Glycobiology 18, 325-330] of mutating Ser(102), which holds a nucleophilic water molecule, and Glu(70), which acts as a catalytic base, producing S102A, S102C, S102D, S102G, S102H, S102T, E70G and E70Q. In all of the mutated enzymes, except S102T, hydrolytic activity towards (GlcNAc)(6) was not detected under the conditions we used. Among the inactive BcChi-A mutants, S102A, S102C, S102G and E70G were found to successfully synthesize (GlcNAc)(4) as a major product from α-(GlcNAc)(2)-F in the presence of (GlcNAc)(2). The S102A mutant showed the greatest glycosynthase activity owing to its enhanced F(-) releasing activity and its suppressed hydrolytic activity. This is the first report on a glycosynthase that employs amino sugar fluoride as a donor substrate.