Analysis of the key active subsites of glycoside hydrolase 13 family members

α-Amylase, pullulanase, neopullulanase, cyclomaltodextrinase (CDase), cyclomaltodextin glucanotransferase (CGTase), etc. are some of the amylolytic enzymes that act on polysaccharides. These enzymes differ from each other with respect to substrate and linkage specificities. These enzymes have been grouped into the GH13 (GH, Glycoside Hydrolase) family in the CAZy database on the basis of similarity in amino acid sequence. Members of this family share three domains viz., A, B, and C, which have several binding subsites to accommodate monomeric units of the polysaccharide substrate. Among these subsites, −2, −1, +1, and +2 subsites are the most critical subsites for catalytic activity. In the present study, the substrate analog-, inhibitor-, or product-bound 3-D structures of 24 members of GH13 family have been analyzed to identify the features of the −2, −1, +1, and +2 subsites shared by all the members for recognition of the common substrate. It is found that neither the number nor the nature of the potential hydrogen bond-forming residues is conserved with the exception of the presence of tyrosine as a stacking residue in the −1 subsite. The relative spatial disposition of the conserved subsite residues are conserved as judged by distance matrices. The backbone of the −2, −1, +1, and +2 subsites does not undergo conformational change for the recognition of the substrate. This analysis suggests that these enzymes recognize their substrate on the basis of shape of the substrate rather than on the basis of specific interactions within the binding site.     

Beta-helical catalytic domains in glycoside hydrolase families 49, 55 and 87: domain architecture,modelling and assignment of catalytic residues

X-ray crystallography and bioinformatics studies reveal a tendency for the right-handed β-helix domain architecture to be associated with carbohydrate binding proteins. Here we demonstrate the presence of catalytic β-helix domains in glycoside hydrolase (GH) families 49, 55 and 87 and provide evidence for their sharing a common evolutionary ancestor with two structurally characterized GH families, numbers 28 and 82. This domain assignment helps assign catalytic residues to each family. Further analysis of domain architecture reveals the association of carbohydrate binding modules with catalytic GH β-helices, as well as an unexpected pair of β-helix domains in GH family 55.     

Thermus thermophilus glycoside hydrolase family 57 branching enzyme: crystal structure, mechanism of action, and products formed

Branching enzyme (EC 2.4.1.18; glycogen branching enzyme; GBE) catalyzes the formation of α1,6-branching points in glycogen. Until recently it was believed that all GBEs belong to glycoside hydrolase family 13 (GH13). Here we describe the cloning and expression of the Thermus thermophilus family GH57-type GBE and report its biochemical properties and crystal structure at 1.35-Å resolution. The enzyme has a central (β/α)₇-fold catalytic domain A with an inserted domain B between β2 and α5 and an α-helix-rich C-terminal domain, which is shown to be essential for substrate binding and catalysis. A maltotriose was modeled in the active site of the enzyme which suggests that there is insufficient space for simultaneously binding of donor and acceptor substrates, and that the donor substrate must be cleaved before acceptor substrate can bind. The biochemical assessment showed that the GH57 GBE possesses about 4% hydrolytic activity with amylose and in vitro forms a glucan product with a novel fine structure, demonstrating that the GH57 GBE is clearly different from the GH13 GBEs characterized to date.     

Three-dimensional structure of a putative non-cellulosomal cohesin module from a Clostridium perfringens family 84 glycoside hydrolase

The genomes of myonecrotic strains of Clostridium perfringens encode a large number of secreted glycoside hydrolases. The activities of these enzymes are consistent with degradation of the mucosal layer of the human gastrointestinal tract, glycosaminoglycans and other cellular glycans found throughout the body. In many cases this is thought to aid in the propagation of the major toxins produced by C. perfringens. One such example is the family 84 glycoside hydrolases, which contains five C. perfringens members (CpGH84A-E), each displaying a unique modular architecture. The smallest and most extensively studied member, CpGH84C, comprises an N-terminal catalytic domain with β-N-acetylglucosaminidase activity, a family 32 carbohydrate-binding module, a family 82 X-module (X82) of unknown function, and a fibronectin type-III-like module. Here we present the structure of the X82 module from CpGH84C, determined by both NMR spectroscopy and X-ray crystallography. CpGH84C X82 adopts a jell-roll fold comprising two β-sheets formed by nine β-strands. CpGH84C X82 displays distant amino acid sequence identity yet close structural similarity to the cohesin modules of cellulolytic anaerobic bacteria. Cohesin modules are responsible for the assembly of numerous hydrolytic enzymes in a cellulose-degrading multi-enzyme complex, termed the cellulosome, through a high-affinity interaction with the calcium-binding dockerin module. A planar surface is located on the face of the CpGH84 X82 structure that corresponds to the dockerin-binding region of cellulolytic cohesin modules and has the approximate dimensions to accommodate a dockerin module. The presence of cohesin-like X82 modules in glycoside hydrolases of C. perfringens is an indication that the formation of novel X82-dockerin mediated multi-enzyme complexes, with potential roles in pathogenesis, is possible.     

The Modular Organisation and Stability of a Thermostable Family 10 Xylanase

The thermophilic marine bacterium Rhodothermus marinus produces a modular family 10 xylanase (Xyn10A). It consists of two N-terminal family 4 carbohydrate binding modules (CBMs) followed by a domain of unknown function (D3), and a catalytic module (CM) flanked by a small fifth domain (D5) at its C-terminus. Several truncated mutants of the enzyme have been produced and characterised with respect to biochemical properties and stability. Multiple calcium binding sites are shown to be present in the two N-terminal CBMs and recent evidence suggests that the third domain of the enzyme also has the ability to bind the same metal ligand. The specific binding of Ca²⁺ was demonstrated to have a pronounced effect on thermostability as shown by differential scanning calorimetry and thermal inactivation studies. Furthermore, deletion mutants of the enzyme were less stable than the full-length enzyme suggesting that module interactions contributed to the stability of the enzyme. Finally, recent evidence indicates that the fifth domain of Xyn10A is a novel type of module mediating cell-attachment.     

The Modular Organisation and Stability of a Thermostable Family 10 Xylanase

The thermophilic marine bacterium Rhodothermus marinus produces a modular family 10 xylanase (Xyn10A). It consists of two N-terminal family 4 carbohydrate binding modules (CBMs) followed by a domain of unknown function (D3), and a catalytic module (CM) flanked by a small fifth domain (D5) at its C-terminus. Several truncated mutants of the enzyme have been produced and characterised with respect to biochemical properties and stability. Multiple calcium binding sites are shown to be present in the two N-terminal CBMs and recent evidence suggests that the third domain of the enzyme also has the ability to bind the same metal ligand. The specific binding of Ca²⁺ was demonstrated to have a pronounced effect on thermostability as shown by differential scanning calorimetry and thermal inactivation studies. Furthermore, deletion mutants of the enzyme were less stable than the full-length enzyme suggesting that module interactions contributed to the stability of the enzyme. Finally, recent evidence indicates that the fifth domain of Xyn10A is a novel type of module mediating cell-attachment.     

Molecular cloning and characterization of a beta-L-Arabinobiosidase in Bifidobacterium longum that belongs to a novel glycoside hydrolase family

Extensin is a glycoprotein that is rich in hydroxyprolines linked to β-L-arabinofuranosides. In this study, we cloned a hypBA2 gene that encodes a novel β-L-arabinobiosidase from Bifidobacterium longum JCM 1217. This enzyme does not have any sequence similarity with other glycoside hydrolase families but has 38-98% identity to hypothetical proteins in Bifidobacterium and Xanthomonas strains. The recombinant enzyme liberated L-arabinofuranose (Araf)-β1,2-Araf disaccharide from carrot extensin, potato lectin, and Araf-β1,2-Araf-β1,2-Araf-β-Hyp (Ara(3)-Hyp) but not Araf-α1,3-Araf-β1,2-Araf-β1,2-Araf-β-Hyp (Ara(4)-Hyp) or Araf-β1,2-Araf-β-Hyp (Ara(2)-Hyp), which indicated that it was specific for unmodified Ara(3)-Hyp substrate. The enzyme also transglycosylated 1-alkanols with retention of the anomeric configuration. This is the first report of an enzyme that hydrolyzes Hyp-linked β-L-arabinofuranosides, which defines a new family of glycoside hydrolases, glycoside hydrolase family 121.     

The nature of the carbohydrate binding module determines the catalytic efficiency of xylanase Z of Clostridium thermocellum

Xylanase Z of Clostridium thermocellum exists as a complex in the cellulosome with N-terminus feruloyl esterase, a carbohydrate binding module (CBM6) and a dockerin domain. To study the role of the binding modules on the activity of XynZ, different variants with the CBM6 attached to the catalytic domain at its C-terminal (XynZ-CB) and N-terminal (XynZ-BC), and the CBM22 attached at N-terminus (XynZ-B′C) were expressed in Escherichia coli at levels around 30% of the total cell proteins. The activities of XynZ-BC, XynZ-CB and XynZ-B′C were 4200, 4180 and 20,700 U μM⁻¹ against birchwood xylan, respectively. Substrate binding studies showed that in case of XynZ-BC and XynZ-CB the substrate birchwood xylan remaining unbound were 51 and 52%, respectively, whereas in the case of XynZ-B′C the substrate remaining unbound was 39% under the assay conditions used. The molecular docking studies showed that the binding site of CBM22 in XynZ-B′C is more exposed and thus available for substrate binding as compared to the tunnel shape binding pocket produced in XynZ-BC and thus hindering the substrate binding. The substrate binding data for the two constructs are in agreement with this explanation.     

Structure of a mannan-specific family 35 carbohydrate-binding module: evidence for significant conformational changes upon ligand binding

Enzymes that digest plant cell wall polysaccharides generally contain non-catalytic, carbohydrate-binding modules (CBMs) that function by attaching the enzyme to the substrate, potentiating catalytic activity. Here, we present the first structure of a family 35 CBM, derived from the Cellvibrio japonicus beta-1,4-mannanase Man5C. The NMR structure has been determined for both the free protein and the protein bound to mannopentaose. The data show that the protein displays a typical beta-jelly-roll fold. Ligand binding is not located on the concave surface of the protein, as occurs in many CBMs that display the jelly-roll fold, but is formed by the loops that link the two beta-sheets of the protein, similar to family 6 CBMs. In contrast to the majority of CBMs, which are generally rigid proteins, CBM35 undergoes significant conformational change upon ligand binding. The curvature of the binding site and the narrow binding cleft are likely to be the main determinants of binding specificity. The predicted solvent exposure of O6 at several subsites provides an explanation for the observed accommodation of decorated mannans. Two of the key aromatic residues in Man5C-CBM35 that interact with mannopentaose are conserved in mannanase-derived CBM35s, which will guide specificity predictions based on the primary sequence of proteins in this CBM family.     

Characterization of a modular enzyme of exo-1,5-alpha-L-arabinofuranosidase and arabinan binding module from Streptomyces avermitilis NBRC14893

A gene encoding an alpha-L: -arabinofuranosidase, designated SaAraf43A, was cloned from Streptomyces avermitilis. The deduced amino acid sequence implies a modular structure consisting of an N-terminal glycoside hydrolase family 43 module and a C-terminal family 42 carbohydrate-binding module (CBM42). The recombinant enzyme showed optimal activity at pH 6.0 and 45 degrees C and was stable over the pH range of 5.0-6.5 at 30 degrees C. The enzyme hydrolyzed p-nitrophenol (PNP)-alpha-L: -arabinofuranoside but did not hydrolyze PNP-alpha-L: -arabinopyranoside, PNP-beta-D: -xylopyranoside, or PNP-beta-D: -galactopyranoside. Debranched 1,5-arabinan was hydrolyzed by the enzyme but arabinoxylan, arabinogalactan, gum arabic, and arabinan were not. Among the synthetic regioisomers of arabinofuranobiosides, only methyl 5-O-alpha-L: -arabinofuranosyl-alpha-L: -arabinofuranoside was hydrolyzed by the enzyme, while methyl 2-O-alpha-L: -arabinofuranosyl-alpha-L: -arabinofuranoside and methyl 3-O-alpha-L: -arabinofuranosyl-alpha-L: -arabinofuranoside were not. These data suggested that the enzyme only cleaves alpha-1,5-linked arabinofuranosyl linkages. The analysis of the hydrolysis product of arabinofuranopentaose suggested that the enzyme releases arabinose in exo-acting manner. These results indicate that the enzyme is definitely an exo-1,5-alpha-L: -arabinofuranosidase. The C-terminal CBM42 did not show any affinity for arabinogalactan and debranched arabinan, although it bound arabinan and arabinoxylan, suggesting that the CBM42 bound to branched arabinofuranosyl residues. Removal of the module decreased the activity of the enzyme with regard to debranched arabinan. The CBM42 plays a role in enhancing the debranched arabinan hydrolytic action of the catalytic module in spite of its preference for binding arabinofuranosyl side chains.     

A secondary xylan-binding site enhances the catalytic activity of a single-domain family 11 glycoside hydrolase

Bacillus circulans xylanase (BcX) is a single-domain family 11 glycoside hydrolase. Using NMR-monitored titrations, we discovered that an inactive variant of this enzyme, E78Q-BcX, bound xylooligosaccharides not only within its pronounced active site (AS) cleft, but also at a distal surface region. Chemical shift perturbation mapping and affinity electrophoresis, combined with mutational studies, identified the xylan-specific secondary binding site (SBS) as a shallow groove lined by Asn, Ser, and Thr residues and with a Trp at one end. The AS and SBS bound short xylooligosaccharides with similar dissociation constants in the millimolar range. However, the on and off-rates to the SBS were at least tenfold faster than those of kon approximately 3x10(5) M(-1) s(-1) and koff approximately 1000 s(-1) measured for xylotetraose to the AS of E78Q-BcX. Consistent with their structural differences, this suggests that a conformational change in the enzyme and/or the substrate is required for association to and dissociation from the deep AS, but not the shallow SBS. In contrast to the independent binding of small xylooligosaccharides, high-affinity binding of soluble and insoluble xylan, as well as xylododecaose, occurred cooperatively to the two sites. This was evidenced by an approximately 100-fold increase in relative Kd values for these ligands upon mutation of the SBS. The SBS also enhances the activity of BcX towards soluble and insoluble xylan through a significant reduction in the Michaelis KM values for these polymeric substrates. This study provides an unexpected example of how a single domain family 11 xylanase overcomes the lack of a carbohydrate-binding module through the use of a secondary binding site to enhance substrate specificity and affinity.     

A novel glycoside hydrolase family 105: the structure of family 105 unsaturated rhamnogalacturonyl hydrolase complexed with a disaccharide in comparison with family 88 enzyme complexed with the disaccharide

YteR, a hypothetical protein with unknown functions, is derived from Bacillus subtilis strain 168 and has an overall structure similar to that of bacterial unsaturated glucuronyl hydrolase (UGL), although it exhibits little amino acid sequence identity with UGL. UGL releases unsaturated glucuronic acid from glycosaminoglycan treated with glycosaminoglycan lyases. The amino acid sequence of YteR shows a significant homology (26% identity) with the hypothetical protein YesR also from B. subtilis strain 168. To clarify the intrinsic functions of YteR and YesR, both proteins were overexpressed in Escherichia coli, purified, and characterized. Based on their gene arrangements in genome and enzyme properties, YteR and YesR were found to constitute a novel enzyme activity, "unsaturated rhamnogalacturonyl hydrolase," classified as new glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) obtained from RG type-I treated with RG lyases and releases an unsaturated galacturonic acid. The crystal structure of YteR complexed with unsaturated chondroitin disaccharide (UGL substrate) was obtained and compared to the structure of UGL complexed with the same disaccharide. The UGL substrate is sterically hindered with the active pocket of YteR. The protruding loop of YteR prevents the UGL substrate from being bound effectively. The most likely candidate catalytic residues for general acid/base are Asp143 in YteR and Asp135 in YesR. This is supported by three-dimensional structural and site-directed mutagenesis studies. These findings provide molecular insights into novel enzyme catalysis and sequential reaction mechanisms involved in RG-I depolymerization by bacteria.     

Structural and biochemical studies of GH family 12 cellulases: improved thermal stability, and ligand complexes

In this review we will describe how we have gathered structural and biochemical information from several homologous cellulases from one class of glycoside hydrolases (GH family 12), and used this information within the framework of a protein-engineering program for the design of new variants of these enzymes. These variants have been characterized to identify some of the positions and the types of mutations in the enzymes that are responsible for some of the biochemical differences in thermal stability and activity between the homologous enzymes. In this process we have solved the three-dimensional structure of four of these homologous GH 12 cellulases: Three fungal enzymes, Humicola grisea Cel12A, Hypocrea jecorina Cel12A and Hypocrea schweinitzii Cel12A, and one bacterial, Streptomyces sp. 11AG8 Cel12A. We have also determined the three-dimensional structures of the two most stable H. jecorina Cel12A variants. In addition, four ligand-complex structures of the wild-type H. grisea Cel12A enzyme have been solved and have made it possible to characterize some of the interactions between substrate and enzyme. The structural and biochemical studies of these related GH 12 enzymes, and their variants, have provided insight on how specific residues contribute to protein thermal stability and enzyme activity. This knowledge can serve as a structural toolbox for the design of Cel12A enzymes with specific properties and features suited to existing or new applications.     

The first structure of a glycoside hydrolase family 61 member, Cel61B from Hypocrea jecorina, at 1.6 A resolution

The glycoside hydrolase (GH) family 61 is a long-recognized, but still recondite, class of proteins, with little known about the activity, mechanism or function of its more than 70 members. The best-studied GH family 61 member, Cel61A of the filamentous fungus Hypocrea jecorina, is known to be an endoglucanase, but it is not clear if this represents the main activity or function of this family in vivo. We present here the first structure for this family, that of Cel61B from H. jecorina. The best-quality crystals were formed in the presence of nickel, and the crystal structure was solved to 1.6 Å resolution using a single-wavelength anomalous dispersion method with nickel as the source of anomalous scatter. Cel61B lacks a carbohydrate-binding module and is a single-domain protein that folds into a twisted β-sandwich. A structure-aided sequence alignment of all GH family 61 proteins identified a highly conserved group of residues on the surface of Cel61B. Within this patch of mostly polar amino acids was a site occupied by the intramolecular nickel hexacoordinately bound in the solved structure. In the Cel61B structure, there is no easily identifiable carbohydrate-binding cleft or pocket or catalytic center of the types normally seen in GHs. A structural comparison search showed that the known structure most similar to Cel61B is that of CBP21 from the Gram-negative soil bacterium Serratia marcescens, a member of the carbohydrate-binding module family 33 proteins. A polar surface patch highly conserved in that structural family has been identified in CBP21 and shown to be involved in chitin binding and in the protein's enhancement of chitinase activities. The analysis of the Cel61B structure is discussed in light of our continuing research to better understand the activities and function of GH family 61.     

A clostridial endo-beta-galactosidase that cleaves both blood group A and B glycotopes: the first member of a new glycoside hydrolase family, GH98

We have isolated an endo-beta-galactosidase designated E-ABase from Clostridium perfringens ATCC 10543 capable of liberating both the A trisaccharide (A-Tri; GalNAcalpha1-->3(Fucalpha1-->2)Gal) and B trisaccharide (B-Tri; Galalpha1-->3(Fucalpha1-->2)Gal) from glycoconjugates containing blood group A and B glycotopes, respectively. We have subsequently cloned the gene (eabC) that encodes E-ABase from this organism. This gene was found to be identical to the CPE0329 gene of C. perfringens strain 13, whose product was labeled as a hypothetical protein (Shimizu, T., Ohtani, K., Hirakawa, H., Ohshima, K., Yamashita, A., Shiba, T., Ogasawara, N., Hattori, M., Kuhara, S., and Hayashi, H. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 996-1001). Since the amino acid sequence of E-ABase does not bear detectable similarity to any of the 97 existing families of glycoside hydrolases, we have proposed to assign this unusual enzyme to a new family, GH98. We also expressed eabC in Escherichia coli BL21(DE3) and obtained 27 mg of fully active recombinant E-ABase from 1 liter of culture. Recombinant E-ABase not only destroyed the blood group A and B antigenicity of human type A and B erythrocytes, but also released A-Tri and B-Tri from blood group A(+)- and B(+)- containing glycoconjugates. The structures of A-Tri and B-Tri liberated from A(+) porcine gastric mucin and B(+) human ovarian cyst glycoprotein were established by NMR spectroscopy. The unique specificity of E-ABase should make it useful for studying the structure and function of blood group A- and B-containing glycoconju-gates as well as for identifying other glycosidases belonging to the new GH98 family.     

Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: insight into the ganglioside binding mechanism

Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinum neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3′-sialyllactose at a resolution of 3.0 Å. In the structure, an electron density derived from the 3′-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.     

Structural insights into the substrate specificity and function of Escherichia coli K12 YgjK, a glucosidase belonging to the glycoside hydrolase family 63

Proteins belonging to the glycoside hydrolase family 63 (GH63) are found in bacteria, archaea, and eukaryotes. Eukaryotic GH63 proteins are processing α-glucosidase I enzymes that hydrolyze an oligosaccharide precursor of eukaryotic N-linked glycoproteins. In contrast, the functions of the bacterial and archaeal GH63 proteins are unclear. Here we determined the crystal structure of a bacterial GH63 enzyme, Escherichia coli K12 YgjK, at 1.78 Å resolution and investigated some properties of the enzyme. YgjK consists of the N-domain and the A-domain, joined by a linker region. The N-domain is composed of 18 antiparallel β-strands and is classified as a super-β-sandwich. The A-domain contains 16 α-helices, 12 of which form an (α/α)₆-barrel; the remaining 4 α-helices are found in an extra structural unit that we designated as the A′-region. YgjK, a member of the glycoside hydrolase clan GH-G, shares structural similarity with glucoamylase (GH15) and chitobiose phosphorylase (GH65), both of which belong to clan GH-L. In crystal structures of YgjK in complex with glucose, mannose, and galactose, all of the glucose, mannose, and galactose units were located in the catalytic cleft. YgjK showed the highest activity for the α-1,3-glucosidic linkage of nigerose, but also hydrolyzed trehalose, kojibiose, and maltooligosaccharides from maltose to maltoheptaose, although the activities were low. These findings suggest that YgjK is a glucosidase with relaxed specificity for sugars.     

Biochemical characterization of Thermotoga maritima endoglucanase Cel74 with and without a carbohydrate binding module (CBM)

The genome of the hyperthermophilic bacterium Thermotoga maritima (Tm) encodes at least eight glycoside hydrolases with putative signal peptides; the biochemical characteristics of seven of these have been reported previously. The eighth, Tm Cel74, is encoded by an open reading frame of 2124 bp corresponding to a polypeptide of 79 kDa with a signal peptide at the amino-terminus. The gene (lacking the signal peptide) encoding Tm Cel74 was expressed as a 77 kDa monomeric polypeptide in Escherichia coli and found to be optimally active at pH 6, 90 degrees C, with a melting temperature of approximately 105 degrees C. The cel74 gene was previously found to be induced during T. maritima growth on a variety of polysaccharides, including barley glucan, carboxymethyl cellulose (CMC), glucomannan, galactomannan and starch. However, while Tm Cel74 was most active towards barley glucan and to a lesser extent CMC, glucomannan and tamarind (xyloglucan), no activity was detected on other glycans, including galactomannan, laminarin and starch. Also, Tm Cel74 did not contain a carbohydrate binding module (CBM), versions of which have been identified in the amino acid sequences of other family 74 enzymes. As such, a CBM associated with a chitinase in another hyperthermophile, Pyrococcus furiosus, was used to create a fusion protein that was active on crystalline cellulose; Tm Cel74 lacked activity on this substrate. Based on the cleavage pattern determined for Tm Cel74 on glucan-based substrates, this enzyme likely initiates recruitment of carbohydrate carbon and energy sources by creating oligosaccharides that are transported into the cell for further processing.