Significance of the 20-kDa subunit of heterodimeric 2-deoxy-scyllo-inosose synthase for the biosynthesis of butirosin antibiotics in Bacillus circulans

A gene (btrC2) encoding the 20-kDa subunit of 2-deoxy-scyllo-inosose (DOI) synthase, a key enzyme in the biosynthesis of 2-deoxystreptamine, was identified from the butirosin-producer Bacillus circulans by reverse genetics. The deduced amino acid sequence of BtrC2 closely resembled that of YaaE of B. subtilis, but the function of the latter has not been known to date. Instead, BtrC2 appeared to show sequence similarity to a certain extent with HisH of B. subtilis, an amidotransferase subunit of imidazole glycerol phosphate synthase. Disruption of btrC2 reduced the growth rate compared with the wild type, and simultaneously antibiotic producing activity was lost. Addition of NH4Cl to the medium complemented only the growth rate of the disruptant, and both the growth rate and antibiotic production were restored by addition of yeast extract. In addition, a heterologous co-expression system of btrC2 with btrC was constructed in Escherichia coli. The simultaneously over-expressed BtrC2 and BtrC constituted a heterodimer, the biochemical features of which resembled those of DOI synthase from B. circulans more than those of the recombinant homodimeric BtrC. Despite the similarity of BtrC2 to HisH the heterodimer showed neither aminotransfer nor amidotransfer activity for 2-deoxy-scyllo-inosose as a substrate. All the observations suggest that BtrC2 is involved not only in the secondary metabolism, but also in the primary metabolism in B. circulans. The function of BtrC2 in the butirosin biosynthesis appears to be indirect, and may be involved in stabilization of DOI synthase and in regulation of its enzyme activity.     

Susceptibility to butirosin and neomycin B in Bacillus circulans, the butirosin-producing organism.

Butirosin, an aminoglycoside antibiotic, is produced by Bacillus circulans B-3312. Experiments using recombined ribosomal and supernatant fractions from this strain and from B. megaterium KM have shown that the ribosome of both are sensitive to butirosin. The aminoglycoside 3'-phosphotransferase present in B. circulans modifies butirosin and neomycin in vitro but confers resistance only to the former in vivo. The phosphotransferase does not modifya detectable amount of extracellular butirosin while mediating resistance to the antibiotic. In vitro, however, the enzyme appears to protect against inhibition by butirosin by inactivating the bulk of the antibiotic in the system. An extrachromosomal element of unknown function has been detected in B. circulans.     

Cytosol aspartate aminotransferase from the chicken heart: three-dimensional structure at 2.8 angstroms resolution and the characteristic conformation of various enzyme forms

The spatial structure of cytosolic chicken aspartate aminotransferase (AAT) has been determined by X-ray crystallographic analysis at 2.8 A resolution. AAT consists of two chemically identical subunits. Each subunit can be subdivided into the large pyridoxal phosphate (PLP) binding domain and the small domain. The two active sites of AAT are situated in deep clefts at the subunit interface. The binding of PLP and 2-oxoglutarate is described. Conformations of the following enzyme forms have been compared by difference Fourier syntheses: the nonliganded PLP-form in phosphate and acetate buffers; the non-liganded pyridoxamine phosphate (PMP) form; complexes of the PLP-form with glutarate and 2-oxoglutarate. Lattice-induced dynamic asymmetry of the dimeric AAT molecules was revealed. In one subunit the small domain is mobile and shifted either toward the active site ("closed" conformation) or in the opposite direction ("open" conformation). The closed conformation is induced by the binding of dicarboxylate anions. In the second subunit the small domain is immobile and shifted toward the active site in all enzyme forms or complexes studied. In this subunit, there occurs a rotation of the PLP ring by approximately 20 degrees toward the substrate site. The rotation is observed when crystals are soaked in 0.6 saturated (NH4)2SO4 solution buffered with 0.3 M potassium phosphate, pH 7.5; it was explained by formation of an external aldimine between PLP and NH3. This aldimine is not formed in the presence of dicarboxylates or acetate. It was inferred that dicarboxylate or acetate anions stabilize the internal PLP-lysine aldimine and prevent its reaction with ammonia. Conversion of AAT from the PLP- to PMP-form is accompanied by rotation of the coenzyme ring by approximately 20 degrees; the rotation occurs in both subunits.     

Expanding substrate specificity of GT-B fold glycosyltransferase via domain swapping and high-throughput screening

Glycosyltransferases (GTs) are crucial enzymes in the biosynthesis and diversification of therapeutically important natural products, and the majority of them belong to the GT-B superfamily, which is composed of separate N- and C-domains that are responsible for the recognition of the sugar acceptor and donor, respectively. In an effort to expand the substrate specificity of GT, a chimeric library with different crossover points was constructed between the N-terminal fragments of kanamycin GT (kanF) and the C-terminal fragments of vancomycin GT (gtfE) genes by incremental truncation method. A plate-based pH color assay was newly developed for the selection of functional domain-swapped GTs, and a mutant (HMT31) with a crossover point (N-kanF-669 bp and 753 bp-gtfE-C) for domain swapping was screened. The most active mutant HMT31 (50 kDa) efficiently catalyzed 2-DOS (aglycone substrate for KanF) glucosylation using dTDP-glucose (glycone substrate for GtfE) with k(cat)/K(m) of 162.8 +/- 0.1 mM(-1) min(-1). Moreover, HMT31 showed improved substrate specificity toward seven more NDP-sugars. This study presents a domain swapping method as a potential means to glycorandomization toward various syntheses of 2-DOS-based aminoglycoside derivatives.     

Crystal structure of diaminopelargonic acid synthase: evolutionary relationships between pyridoxal-5'-phosphate-dependent enzymes.

The three-dimensional structure of diaminopelargonic acid synthase, a vitamin B6-dependent enzyme in the pathway of the biosynthesis of biotin, has been determined to 1.8 A resolution by X-ray crystallography. The structure was solved by multi-wavelength anomalous diffraction techniques using a crystal derivatized with mercury ions. The protein model has been refined to a crystallographic R -value of 17.5% (R -free 22.6%). Each enzyme subunit consists of two domains, a large domain (residues 50-329) containing a seven-stranded predominantly parallel beta-sheet, surrounded by alpha-helices, and a small domain comprising residues 1-49 and 330-429. Two subunits, related by a non-crystallographic dyad in the crystals, form the homodimeric molecule, which contains two equal active sites. Pyridoxal-5'-phosphate is bound in a cleft formed by both domains of one subunit and the large domain of the second subunit. The cofactor is anchored to the enzyme by a covalent linkage to the side-chain of the invariant residue Lys274. The phosphate group interacts with main-chain nitrogen atoms and the side-chain of Ser113, located at the N terminus of an alpha-helix. The pyridine nitrogen forms a hydrogen bond to the side-chain of the invariant residue Asp245. Electron density corresponding to a metal ion, most likely Na(+), was found in a tight turn at the surface of the enzyme. Structure analysis reveals that diaminopelargonic acid synthase belongs to the family of vitamin B6-dependent aminotransferases with the same fold as originally observed in aspartate aminotransferase. A multiple structure alignment of enzymes in this family indicated that they form at least six different subclasses. Striking differences in the fold of the N-terminal part of the polypeptide chain are one of the hallmarks of these subclasses. Diaminopelargonic acid synthase is a member of the aminotransferase subclass III. From the structure of the non-productive complex of the holoenzyme with the substrate 7-keto-8-aminopelargonic acid the location of the active site and residues involved in substrate binding have been identified.     

Toward understanding the mechanism of the complex cyclization reaction catalyzed by imidazole glycerolphosphate synthase: crystal structures of a ternary complex and the free enzyme

Imidazole glycerol phosphate synthase catalyzes formation of the imidazole ring in histidine biosynthesis. The enzyme is also a glutamine amidotransferase, which produces ammonia in a glutaminase active site and channels it through a 30-A internal tunnel to a cyclase active site. Glutaminase activity is impaired in the resting enzyme, and stimulated by substrate binding in the cyclase active site. The signaling mechanism was investigated in the crystal structure of a ternary complex in which the glutaminase active site was inactivated by a glutamine analogue and the unstable cyclase substrate was cryo-trapped in the active site. The orientation of N(1)-(5'-phosphoribulosyl)-formimino-5-aminoimidazole-4-carboxamide ribonucleotide in the cyclase active site implicates one side of the cyclase domain in signaling to the glutaminase domain. This side of the cyclase domain contains the interdomain hinge. Two interdomain hydrogen bonds, which do not exist in more open forms of the enzyme, are proposed as molecular signals. One hydrogen bond connects the cyclase domain to the substrate analogue in the glutaminase active site. The second hydrogen bond connects to a peptide that forms an oxyanion hole for stabilization of transient negative charge during glutamine hydrolysis. Peptide rearrangement induced by a fully closed domain interface is proposed to activate the glutaminase by unblocking the oxyanion hole. This interpretation is consistent with biochemical results [Myers, R. S., et al., (2003) Biochemistry 42, 7013-7022, the accompanying paper in this issue] and with structures of the free enzyme and a binary complex with a second glutamine analogue.     

Crystal structure of pyridoxamine-pyruvate aminotransferase from Mesorhizobium loti MAFF303099

Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a pyridoxal 5'-phosphate-independent aminotransferase and catalyzes reversible transamination between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P4(3)2(1)2 and was refined to an R factor of 15.6% (R(free) = 20.6%) at 2.0 A resolution. In addition, the structures of PPAT in complexes with pyridoxamine, pyridoxal, and pyridoxyl-L-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (R(free) = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0 A resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven beta-sheets and eight alpha-helices, and a smaller C-terminal domain, consisting of three beta-sheets and four alpha-helices. The substrate pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The alpha-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of pyridoxal 5'-phosphate and made PPAT specific to pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.     

Assessing the structural conservation of protein pockets to study functional and allosteric sites: implications for drug discovery

Background

Nonribosomal peptide synthetases (NRPSs) are multienzymatic, multidomain megasynthases involved in the biosynthesis of pharmaceutically important nonribosomal peptides. The peptaibol synthetase from Trichoderma virens (TPS) is an important member of the NRPS family that exhibits antifungal properties. The majority of the NRPSs terminate peptide synthesis with the thioesterase (TE) domain, which either hydrolyzes the thioester linkage, releasing the free peptic acid, or catalyzes the intramolecular macrocyclization to produce a macrolactone product. TPS is an important NRPS that does not encompass a TE domain, but rather a reductase domain (R domain) to release the mature peptide product reductively with the aid of a NADPH cofactor. However, the catalytic mechanism of the reductase domain has not yet been elucidated.

Results

We present here a three-dimensional (3D) model of the reductase domain based on the crystal structure of vestitone reductase (VR). VR belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and is responsible for the nicotinamide dinucleotide phosphate (NADPH)-dependent reduction of the substrate into its corresponding secondary alcohol product. The binding sites of the probable linear substrates, alamethicin, trichotoxin, antiamoebin I, chrysopermin C and gramicidin, were identified within the modeled R domain using multiple docking approaches. The docking results of the ligand in the active site of the R domain showed that reductase side chains have a high affinity towards ligand binding, while the thioester oxygen of each substrate forms a hydrogen bond with the OH group of Tyr176 and the thiol group of the substrate is closer to the Glu220. The modeling and docking studies revealed the reaction mechanism of reduction of thioester into a primary alcohol.

Conclusion

Peptaibol biosynthesis incorporates a single R domain, which appears to catalyze the four-electron reduction reaction of a peptidyl carrier protein (PCP)-bound peptide to its corresponding primary alcohol. Analysis of R domains present in the non-redundant (nr) database of the NCBI showed that the R domain always resides in the last NRPS module and is involved in either a two or four-electron reduction reaction.     

Study of possible biological transformation of kanamycin to amikacin]

For transformation of kanamycin A (Km) to amikacin (Ak) with acylating enzymes from B. circulans, a culture producing butirosin (Btn), cellular and acellular conversion systems and methods for chemical and biological identification of Km, Ak and Btn were developed. The level of conversion of Km to Ak in vivo and in vitro did not exceed 2 per cent.     

Crystal structure of carbapenam synthetase (CarA)

Carbapenam synthetase (CarA) is an ATP/Mg2+-dependent enzyme that catalyzes formation of the beta-lactam ring in (5R)-carbapenem-3-carboxylic acid biosynthesis. CarA is homologous to beta-lactam synthetase (beta-LS), which is involved in clavulanic acid biosynthesis. The catalytic cycles of CarA and beta-LS mediate substrate adenylation followed by beta-lactamization via a tetrahedral intermediate or transition state. Another member of this family of ATP/Mg2+-dependent enzymes, asparagine synthetase (AS-B), catalyzes intermolecular, rather than intramolecular, amide bond formation in asparagine biosynthesis. The crystal structures of apo-CarA and CarA complexed with the substrate (2S,5S)-5-carboxymethylproline (CMPr), ATP analog alpha,beta-methyleneadenosine 5'-triphosphate (AMP-CPP), and a single Mg2+ ion have been determined. CarA forms a tetramer. Each monomer resembles beta-LS and AS-B in overall fold, but key differences are observed. The N-terminal domain lacks the glutaminase active site found in AS-B, and an extended loop region not observed in beta-LS or AS-B is present. Comparison of the C-terminal synthetase active site to that in beta-LS reveals that the ATP binding site is highly conserved. By contrast, variations in the substrate binding pocket reflect the different substrates of the two enzymes. The Mg2+ coordination is also different. Several key residues in the active site are conserved between CarA and beta-LS, supporting proposed roles in beta-lactam formation. These data provide further insight into the structures of this class of enzymes and suggest that CarA might be a versatile target for protein engineering experiments aimed at developing improved production methods and new carbapenem antibiotics.     

Role of glutamate 243 in the active site of 2-deoxy-scyllo-inosose synthase from Bacillus circulans

2-Deoxy-scyllo-inosose (DOI) synthase is involved in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics and catalyzes the carbocyclic formation from d-glucose-6-phosphate (G-6-P) into DOI. The reaction mechanism is proposed to be similar to that of dehydroquinate (DHQ) synthase in the shikimate pathway, and includes oxidation of C-4, beta-elimination of phosphate, reduction of C-4, ring opening, and intramolecular aldol cyclization. To investigate the reaction mechanism of DOI synthase, site-directed mutational analysis of three presumable catalytically important amino acids of DOI synthase derived from the butirosin producer Bacillus circulans (BtrC) was carried out. Steady state and pre-steady state kinetic analysis suggested that E243 of BtrC is catalytically involved in the phosphate elimination step. Further analysis of the mutant E243Q of BtrC using substrate analogue, glucose-6-phosphonate, clearly confirmed that E243 was responsible to abstract a proton at C-5 in G-6-P and set off phosphate elimination. This glutamate residue is completely conserved in all DOI synthases identified so far and the corresponding amino acid of DHQ synthase is completely conserved as asparagine. Therefore, this characteristic glutamate residue of DOI synthase is a key determinant to distinguish the reaction mechanism between DOI synthase and DHQ synthase as well as primary sequence.     

Crystal structures of unbound and aminooxyacetate-bound Escherichia coli gamma-aminobutyrate aminotransferase

The X-ray crystal structures of Escherichia coli gamma-aminobutyrate aminotransferase unbound and bound to the inhibitor aminooxyacetate are reported. The enzyme crystallizes from ammonium sulfate solutions in the P3(2)21 space group with a tetramer in the asymmetric unit. Diffraction data were collected to 2.4 A resolution for the unliganded enzyme and 1.9 A resolution for the aminooxyacetate complex. The overall structure of the enzyme is similar to those of other aminotransferase subgroup II enzymes. The ability of gamma-aminobutyrate aminotransferase to act on primary amine substrates (gamma-aminobutyrate) in the first half-reaction and alpha-amino acids in the second is proposed to be enabled by the presence of Glu211, whose side chain carboxylate alternates between interactions with Arg398 in the primary amine half-reaction and an alternative binding site in the alpha-amino acid half-reaction, in which Arg398 binds the substrate alpha-carboxylate. The specificity for a carboxylate group on the substrate side chain is due primarily to the presence of Arg141, but also requires substantial local main chain rearrangements relative to the structurally homologous enzyme dialkylglycine decarboxylase, which is specific for small alkyl side chains. No iron-sulfur cluster is found in the bacterial enzyme as was found in the pig enzyme [Storici, P., De Biase, D., Bossa, F., Bruno, S., Mozzarelli, A., Peneff, C., Silverman, R. B., and Schirmer, T. (2004) J. Biol. Chem. 279, 363-73.]. The binding of aminooxyacetate causes remarkably small changes in the active site structure, and no large domain movements are observed. Active site structure comparisons with pig gamma-aminobutyrate aminotransferase and dialkylglycine decarboxylase are discussed.     

Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

Homocitrate synthase (HCS) catalyzes the first and committed step in lysine biosynthesis in many fungi and certain Archaea and is a potential target for antifungal drugs. Here we report the crystal structure of the HCS apoenzyme from Schizosaccharomyces pombe and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate (2-OG). The structures reveal that HCS forms an intertwined homodimer stabilized by domain-swapping between the N- and C-terminal domains of each monomer. The N-terminal catalytic domain is composed of a TIM barrel fold in which 2-OG binds via hydrogen bonds and coordination to the active site divalent metal ion, whereas the C-terminal domain is composed of mixed α/β topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain occludes the entrance to the active site of the neighboring monomer, whereas in the second 2-OG complex the lid is disordered, suggesting that it regulates substrate access to the active site through its apparent flexibility. Mutations of the active site residues involved in 2-OG binding or implicated in acid-base catalysis impair or abolish activity in vitro and in vivo. Together, these results yield new insights into the structure and catalytic mechanism of HCSs and furnish a platform for developing HCS-selective inhibitors.     

Crystal structure of vestitone reductase from alfalfa (Medicago sativa L.)

Isoflavonoids are commonly found in leguminous plants, where they play important roles in plant defense and have significant health benefits for animals and humans. Vestitone reductase catalyzes a stereospecific NADPH-dependent reduction of (3R)-vestitone in the biosynthesis of the antimicrobial isoflavonoid phytoalexin medicarpin. The crystal structure of alfalfa (Medicago sativa L.) vestitone reductase has been determined at 1.4 A resolution. The structure contains a classic Rossmann fold domain in the N terminus and a small C-terminal domain. Sequence and structural analysis showed that vestitone reductase is a member of the short-chain dehydrogenase/reductase (SDR) superfamily despite the low levels of sequence identity, and the prominent structural differences from other SDR enzymes with known structures. The putative binding sites for the co-factor NADPH and the substrate (3R)-vestitone were defined and located in a large cleft formed between the N and C-terminal domains of enzyme. Potential key residues for enzyme activity were also identified, including the catalytic triad Ser129-Tyr164-Lys168. A molecular docking study showed that (3R)-vestitone, but not the (3S) isomer, forms favored interactions with the co-factor and catalytic triad, thus providing an explanation for the enzyme's strict substrate stereo-specificity.     

Crystal structure of Saccharomyces cerevisiae Aro8, a putative α‐aminoadipate aminotransferase

α‐Aminoadipate aminotransferase (AAA‐AT) catalyzes the amination of 2‐oxoadipate to α‐aminoadipate in the fourth step of the α‐aminoadipate pathway of lysine biosynthesis in fungi. The aromatic aminotransferase Aro8 has recently been identified as an AAA‐AT in Saccharomyces cerevisiae. This enzyme displays broad substrate selectivity, utilizing several amino acids and 2‐oxo acids as substrates. Here we report the 1.91Å resolution crystal structure of Aro8 and compare it to AAA‐AT LysN from Thermus thermophilus and human kynurenine aminotransferase II. Inspection of the active site of Aro8 reveals asymmetric cofactor binding with lysine‐pyridoxal‐5‐phosphate bound within the active site of one subunit in the Aro8 homodimer and pyridoxamine phosphate and a HEPES molecule bound to the other subunit. The HEPES buffer molecule binds within the substrate‐binding site of Aro8, yielding insights into the mechanism by which it recognizes multiple substrates and how this recognition differs from other AAA‐AT/kynurenine aminotransferases.     

The structural basis for allosteric inhibition of a threonine-sensitive aspartokinase

The commitment step to the aspartate pathway of amino acid biosynthesis is the phosphorylation of aspartic acid catalyzed by aspartokinase (AK). Most microorganisms and plants have multiple forms of this enzyme, and many of these isofunctional enzymes are subject to feedback regulation by the end products of the pathway. However, the archeal species Methanococcus jannaschii has only a single, monofunctional form of AK. The substrate l-aspartate binds to this recombinant enzyme in two different orientations, providing the first structural evidence supporting the relaxed regiospecificity previously observed with several alternative substrates of Escherichia coli AK ( Angeles, T. S., Hunsley, J. R., and Viola, R. E. (1992) Biochemistry 31, 799-805 ). Binding of the nucleotide substrate triggers significant domain movements that result in a more compact quaternary structure. In contrast, the highly cooperative binding of the allosteric regulator l-threonine to multiple sites on this dimer of dimers leads to an open enzyme structure. A comparison of these structures supports a mechanism for allosteric regulation in which the domain movements induced by threonine binding causes displacement of the substrates from the enzyme, resulting in a relaxed, inactive conformation.     

Structural and biochemical basis for the substrate specificity of Pad-1, an indole-3-pyruvic acid aminotransferase in auxin homeostasis

Phytohormone indole-3-acetic acid (IAA) plays a vital role in regulating plant growth and development. Tryptophan-dependent IAA biosynthesis participates in IAA homeostasis by producing IAA via two sequential reactions, which involve a conversion of tryptophan to indole-3-pyruvic acid (IPyA) by tryptophan aminotransferase (TAA1) followed by the irreversible formation of IAA in the second reaction. Pad-1 from Solanaceae plants regulates IAA levels by catalyzing a reverse reaction of the first step of IAA biosynthesis. Pad-1 is a pyridoxal phosphate (PLP)-dependent aminotransferase, with IPyA as the amino acceptor and l-glutamine as the amino donor. Currently, the structural and functional basis for the substrate specificity of Pad-1 remains poorly understood. In this study, we carried out structural and kinetic analyses of Pad-1 from Solanum melongena. Pad-1 is a homodimeric enzyme, with coenzyme PLP present between a central large α/β domain and a protruding small domain. The active site of Pad-1 includes a vacancy near the phosphate group (P-side) and the 3′-O (O-side) of PLP. These features are distinct from those of TAA1, which is homologous in an overall structure with Pad-1 but includes only the P-side region in the active site. Kinetic analysis suggests that P-side residues constitute a binding pocket for l-glutamine, and O-side residues of Phe124 and Ile350 are involved in the binding of IPyA. These studies illuminate distinct differences in the active site between Pad-1 and TAA1, and provide structural and functional insights into the substrate specificity of Pad-1.     

Crystal structure of chitosanase from Bacillus circulans MH-K1 at 1.6-A resolution and its substrate recognition mechanism.

Chitosanase from Bacillus circulans MH-K1 is a 29-kDa extracellular protein composed of 259 amino acids. The crystal structure of chitosanase from B. circulans MH-K1 has been determined by multiwavelength anomalous diffraction method and refined to crystallographic R = 19.2% (R(free) = 23.5%) for the diffraction data at 1.6-A resolution collected by synchrotron radiation. The enzyme has two globular upper and lower domains, which generate the active site cleft for the substrate binding. The overall molecular folding is similar to chitosanase from Streptomyces sp. N174, although there is only 20% identity at the amino acid sequence level between both chitosanases. However, there are three regions in which the topology is remarkably different. In addition, the disulfide bridge between Cys(50) and Cys(124) joins the beta1 strand and the alpha7 helix, which is not conserved among other chitosanases. The orientation of two backbone helices, which connect the two domains, is also different and is responsible for the differences in size and shape of the active site cleft in these two chitosanases. This structural difference in the active site cleft is the reason why the enzymes specifically recognize different substrates and catalyze different types of chitosan degradation.