Dissecting structural basis of the unique substrate selectivity of human enteropeptidase catalytic subunit

Enteropeptidase is a key enzyme in the digestion system of higher animals. It initiates enzymatic cascade cleaving trypsinogen activation peptide after a unique sequence DDDDK. Recently, we have found specific activity of human enteropeptidase catalytic subunit (L-HEP) being significantly higher than that of its bovine ortholog (L-BEP). Moreover, we have discovered that L-HEP hydrolyzed several nonspecific peptidic substrates. In this work, we aimed to further characterize species-specific enteropeptidase activities and to reveal their structural basis. First, we compared hydrolysis of peptides and proteins lacking DDDDK sequence by L-HEP and L-BEP. In each case human enzyme was more efficient, with the highest hydrolysis rate observed for substrates with a large hydrophobic residue in P2-position. Computer modeling suggested enzyme exosite residues 96 (Arg in L-HEP, Lys in L-BEP) and 219 (Lys in L-HEP, Gln in L-BEP) to be responsible for these differences in enteropeptidase catalytic activity. Indeed, human-to-bovine mutations Arg96Lys, Lys219Gln shifted catalytic properties of L-HEP toward those of L-BEP. This effect was amplified in case of the double mutation Arg96Lys/Lys219Gln, but still did not cover the full difference in catalytic activities of human and bovine enzymes. To find a missing link, we studied monopeptide benzyl-arginine-β-naphthylamide hydrolysis. L-HEP catalyzed it with an order lower K _m than L-BEP, suggesting the monopeptide-binding S1 site input into catalytic distinction between two enteropeptidase species. Together, our findings suggest structural basis of the unique catalytic properties of human enteropeptidase and instigate further studies of its tentative physiological and pathological roles.     

Enhancement of the substrate specificity of D-amino acid oxidase based on tunnel-pocket engineering

D -Amino acid oxidase (DAAO) selectively catalyzes the oxidative deamination of  D -amino acids, making it one of the most promising routes for synthesizing optically pure  L -amino acids, including  L -phosphinothricin ( L -PPT), a chiral herbicide with significant market potential. However, the native DAAOs that have been reported have low activity against unnatural acid substrate  D -PPT. Herein, we designed and screened a DAAO from Rhodotorula taiwanensis (RtwDAAO), and improved its catalytic potential toward  D -PPT through protein engineering. A semirational design approach was employed to create a mutation library based on the tunnel-pocket engineering. After three rounds of iterative saturation mutagenesis, the optimal variant M_3rd-SHVG was obtained, exhibiting a >2000-fold increase in relative activity. The kinetic parameters showed that M_3rd-SHVG improved the substrate binding affinity and turnover number. This is the optimal parameter reported so far. Further, molecular dynamics simulation revealed that the M_3rd-SHVG reshapes the tunnel-pocket and corrects the direction of enzyme–substrate binding, allowing efficiently catalyze unnatural substrates. Our strategy demonstrates that the redesign of tunnel-pockets is effective in improving the activity and kinetic efficiency of DAAO, which provides a valuable reference for enzymatic catalysis. With the M_3rd-SHVG as biocatalyst, 500 mM D, L -PPT was completely converted and the yield reached 98%. The results laid the foundation for further industrial production.     

Insights into the anthrax lethal factor–substrate interaction and selectivity using docking and molecular dynamics simulations

The anthrax toxin of the bacterium Bacillus anthracis consists of three distinct proteins, one of which is the anthrax lethal factor (LF). LF is a gluzincin Zn‐dependent, highly specific metalloprotease with a molecular mass of ～90 kDa that cleaves most isoforms of the family of mitogen‐activated protein kinase kinases (MEKs/MKKs) close to their amino termini, resulting in the inhibition of one or more signaling pathways. Previous studies on the crystal structures of uncomplexed LF and LF complexed with the substrate MEK2 or a MKK‐based synthetic peptide provided structure‐activity correlations and the basis for the rational design of efficient inhibitors. However, in the crystallographic structures, the substrate peptide was not properly oriented in the active site because of the absence of the catalytic zinc atom. In the current study, docking and molecular dynamics calculations were employed to examine the LF‐MEK/MKK interaction along the catalytic channel up to a distance of 20 Å from the zinc atom. This residue‐specific view of the enzyme‐substrate interaction provides valuable information about: (i) the substrate selectivity of LF and its inactivation of MEKs/MKKs (an issue highly important not only to anthrax infection but also to the pathogenesis of cancer), and (ii) the discovery of new, previously unexploited, hot‐spots of the LF catalytic channel that are important in the enzyme/substrate binding and interaction.     

Nucleophile selectivity in the acyl transfer reaction of a designed enzyme

The acyl transfer reaction of S-glutathionyl benzoate (GSB) is catalyzed by a rationally designed mutant of human glutathione transferase A1-1, A216H. The catalyzed reaction proceeds via the formation of an acyl intermediate and has been studied in the presence of nitrogen, oxygen, and sulfur nucleophiles to determine the selectivity with regards to nucleophile structure. Methanol was previously shown to react with the acyl intermediate and form the corresponding ester, methylbenzoate, under a significant rate enhancement. In the present investigation, the dependence on nucleophile structure and reactivity has been investigated. Ethane thiol gave rise to a larger rate enhancement in the enzyme-catalyzed reaction than ethanol, whereas ethylamine did not increase the reaction rate. The reactivities toward the acyl intermediate of primary and secondary alcohols with similar pKa values depended on the structure of the aliphatic chain, and 1-propanol was the most efficient alcohol. The reactivity of the oxygen nucleophiles was also found to depend strongly on pKa as 2,2,2-trifluoroethanol, with a pKa of 12.4, was the most efficient nucleophile of all that were tested. Saturation kinetics was observed in the case of 1-propanol, indicating a second binding site in the active site of A216H. The nucleophile selectivity of A216H provides the knowledge base needed for the further reengineering of A216H towards alternative substrate specificities.     

AA10家族裂解多糖单加氧酶的模块化组成及底物选择性的研究进展

AA10家族裂解多糖单加氧酶(lytic polysaccharide monooxygenases, LPMOs)主要分布于细菌中,因其具有催化纤维素和几丁质等结晶多糖氧化降解的特性,在工业生物质转化过程中具有极强的应用潜力,从而受到广泛关注。然而,AA10家族不同LPMOs作用的底物种类及氧化位点和氧化产物也不尽相同,LPMOs的结构与组成对其底物选择性的影响机制有待进一步探究。因此,本文综述了AA10家族LPMOs的模块化结构组成及其催化机制,梳理了AA10家族LPMOs的底物谱,系统总结了AA10家族LPMOs的结构、关键作用残基及多模块组合对底物选择性影响的最新进展,并展望了LPMOs在生物质转化和生物燃料工业中广阔的应用前景。     

Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability

Pseudomonas aeruginosa Ph1 is a mutant strain derived from strain AI3. The strain AI3 is able to use acetanilide as a carbon source through a mutation (T103I) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thr103Ile and Trp138Gly) by direct sequencing of PCR-amplified mutant gene from strain Ph1 and confirmed by sequencing the cloned PCR-amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wild-type and mutant amidase genes (W138G, T103I-W138G, and T103I) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed several differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T103I) amidase acted only on p-nitrophenylacetamide and phenylacetamide. On the other hand, the T103I enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermostable than the wild-type enzyme, as the mutant (W138G and W138G-T103I) enzymes exhibited t _1/2 values of 7.0 and 1.5 min at 55°C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instabiliby due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its M _r value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.     

Enhancement of apparent substrate selectivity of proteinase K encapsulated in liposomes through a cholate-induced alteration of the bilayer permeability

Proteinase K-containing liposomes with highly selective membrane permeability properties were prepared. The selectivity obtained was with respect to the two substrate molecules added to the external aqueous phase of the liposomes: acetyl-L-Ala-Ala-Ala-p-nitroanilide (Ac-AAA-pNA) and succinyl-L-Ala-Ala-Ala-p-nitroanilide (Suc-AAA-pNA). The liposome-forming lipid used was POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and modulation of the membrane permeability was achieved using the detergent cholate. Proteinase K-containing mixed liposomes (PKCL) were prepared by adding cholate to preformed proteinase K-containing POPC liposomes (PKL) at a defined effective cholate/POPC molar ratio in the liposomal bilayer membrane R(e). Proteinase K was kept inside PKCL with a negligible amount of leakage into the bulk aqueous phase at R(e) < or = 0.30. At higher R(e), leakage of proteinase K was pronounced, even under conditions where POPC/cholate mixed liposomes seemed to be still intact (0.30 < R(e) < or = 0.39). At R(e) < or = 0.30, the reactivity of proteinase K in the PKCL measured with the externally added substrate Ac-AAA-pNA increased with increasing R(e), while the reactivity measured with Suc-AAA-pNA remained low, regardless of the R(e) value. This showed that externally added Ac-AAA-pNA molecules permeated the liposomal membrane more easily than Suc-AAA-pNA by modulating the membrane with cholate. Consequently, Ac-AAA-pNA was hydrolyzed in PKCL with considerably higher apparent substrate selectivity in comparison with the cases of proteinase K in PKL and free proteinase K (without liposomal encapsulation). The results obtained clearly demonstrate that the prepared PKCL can be utilized as a kind of nano-scaled bioreactor system which can take up a particular target substrate with high apparent substrate selectively from the external phase of the liposomes. Inside the liposomes, the target substrate is then converted into the corresponding products.     

Combining substrate dynamics, binding statistics, and energy barriers to rationalize regioselective hydroxylation of octane and lauric acid by CYP102A1 and mutants

Hydroxylations of octane and lauric acid by Cytochrome P450-BM3 (CYP102A1) wild-type and three active site mutants--F87A, L188Q/A74G, and F87V/L188Q/A74G--were rationalized using a combination of substrate orientation from docking, substrate binding statistics from molecular dynamics simulations, and barrier energies for hydrogen atom abstraction from quantum mechanical calculations. Wild-type BM3 typically hydroxylates medium- to long-chain fatty acids on subterminal (omega-1, omega-2, omega-3) but not the terminal (omega) positions. The known carboxylic anchoring site Y51/R47 for lauric acid, and hydrophobic interactions and steric exclusion, mainly by F87, for octane as well as lauric acid, play a role in the binding modes of the substrates. Electrostatic interactions between the protein and the substrate strongly modulate the substrate's regiodependent activation barriers. A combination of the binding statistics and the activation barriers of hydrogen-atom abstraction in the substrates is proposed to determine the product formation. Trends observed in experimental product formation for octane and lauric acid by wild-type BM3 and the three active site mutants were qualitatively explained. It is concluded that the combination of substrate binding statistics and hydrogen-atom abstraction barrier energies is a valuable tool to rationalize substrate binding and product formation and constitutes an important step toward prediction of product ratios.     

Substrate selectivity of lipases in the esterification of oleic acid, linoleic acid, linolenic acid and their all-trans-isomers and in the transesterification of cis/trans-isomers of linoleic acid methyl ester

The substrate selectivity of several microbial lipases has been examined in the esterification of oleic acid, linoleic acid, linolenic acid and their all-trans-isomers and in the alcoholysis of isomeric linoleic acid methyl esters with n-butanol. Lipases from Candida cylindracea and Mucor miehei preferred fatty acids and methyl esters with a (first) cis double bond in 9-position, while Chirazyme L-5, a Candida antarctica lipase A, had a preference for trans-9 unsaturated substrates.     

Modifying the oligomeric state of cyclic amidase and its effect on enzymatic catalysis

A group of cyclic amidases, including hydantoinase, allantoinase, dihydropyrimidinase, and dihydroorotase, catalyze the reversible hydrolysis of cyclic ureides, such as 5-monosubstituted hydantoins and dihydropyrimidines. These four enzymes carry hydrophobic patches to form dimers. With the exception of dihydroorotase, these enzymes are further dimerized to form tetramers by hydrophobic interactions. This leads us to speculate that the hydrophobic interaction domain may be a significant factor in the catalytic property of these oligomeric cyclic amidases, for which activities are not allosterically regulated. We generated a dimeric D-hydantoinase by mutating five residues in the hydrophobic alpha-helical interface of a tetramer and analyzed the kinetic properties of the dimeric form of D-hydantoinase. The specific activity of the dimeric D-hydantoinase corresponds to 5.3% of the activity of tetrameric D-hydantoinase. This low specific activity of the dimeric D-hydantoinase indicates that the dimeric interaction to form a tetramer has a significant effect on the catalytic activity of this non-allosteric tetramer.     

Role of sequence evolution and conformational dynamics in the substrate specificity and oligomerization mode of thymidylate kinases

Thymidylate kinase (TMK) is a key enzyme for the synthesis of DNA, making it an important target for the development of anticancer, antibacterial, and antiparasitic drugs. TMK homologs exhibit significant variations in sequence, residue conformation, substrate specificity, and oligomerization mode. However, the influence of sequence evolution and conformational dynamics on its quaternary structure and function has not been studied before. Based on extensive sequence and structure analyses, our study detected several non-conserved residues which are linked by co-evolution and are implicated in the observed variations in flexibility, oligomeric assembly, and substrate specificity among the homologs. These lead to differences in the pattern of interactions at the active site in TMKs of different specificity. The method was further tested on TMK from Sulfolobus tokodaii (StTMK) which has substantial differences in sequence and structure compared to other TMKs. Our analyses pointed to a more flexible dTMP-binding site in StTMK compared to the other homologs. Binding assays proved that the protein can accommodate both purine and pyrimidine nucleotides at the dTMP binding site with comparable affinity. Additionally, the residues responsible for the narrow specificity of Brugia malayi TMK, whose three-dimensional structure is unavailable, were detected. Our study provides a residue-level understanding of the differences observed among TMK homologs in previous experiments. It also illustrates the correlation among sequence evolution, conformational dynamics, oligomerization mode, and substrate recognition in TMKs and detects co-evolving residues that affect binding, which should be taken into account while designing novel inhibitors.     

In silico design and molecular basis for the selectivity of Olinone toward the first over the second bromodomain of BRD4

Bromodomains (BrDs), a conserved structural module in chromatin-associated proteins, are well known for recognizing ε-N-acetyl lysine residues on histones. One of the most relevant BrDs is BRD4, a tandem BrD containing protein (BrD1 and BrD2) that plays a critical role in numerous diseases including cancer. Growing evidence shows that the two BrDs of BRD4 have different biological functions; hence selective ligands that can be used to study their functions are of great interest. Here, as a follow-up of our previous work, we first provide a detailed characterization study of the in silico rational design of Olinone as part of a series of five tetrahydropyrido indole-based compounds as BRD4 BrD1 inhibitors. Additionally, we investigated the molecular basis for Olinone's selective recognition by BrD1 over BrD2. Molecular dynamics simulations, free energy calculations, and conformational analyses of the apo-BRD4-BrD1|2 and BRD4-BrD1|2/Olinone complexes showed that Olinone's selectivity is facilitated by five key residues: Leu92 in BrD1|385 in BrD2 of ZA loop, Asn140|433, Asp144|His437 and Asp145|Glu438 of BC loop, and Ile146|Val49 of helix C. Furthermore, the difference in hydrogen bonds number and in mobility of the ZA and BC loops of the acetyl-lysine binding site between BRD4 BrD1/Olinone and BrD2/Olinone complexes also contribute to the difference in Olinone's binding affinity and selectivity toward BrD1 over BrD2. Altogether, our computer-aided molecular design techniques can effectively guide the development of small-molecule BRD4 BrD1 inhibitors, explain their selectivity origin, and further open doors to the design of new therapeutically improved derivatives.     

Effect of water activity and immobilization on fatty acid selectivity for esterification reactions mediated by lipases

The effect of water activity (a(w)) and immobilization on fatty acid (FA) selectivity of Burkholderia (formerly Pseudomonas) cepacia, Rhizomucor miehei, Candida antarctica (type B), and Candida rugosa lipases in esterification reactions was determined. Studies were based on measuring ester formation in multicompetitive reaction mixtures containing either the homologous series of even carbon number n-chain saturated FA (C4-C18) or a series of n-chain (un)saturated FA (C18:X, where X = 0-3 double bonds) as cosubstrates with 1,3-propanediol in ter-butyl methyl ether at a(w) of 0.19, 0.69, and 0.90. Activity and FA selectively patterns were similar for free and Celite-adsorbed lipases in response to changes in a(w'), although specific effects were observed for selectivity of B. cepacia and C. rugosa lipases toward C16 and C4/C6 FA, respectively. Also, selectivity toward unsaturated C18:X FA as a group was modulated by changes in a(w) for three of the four lipase studied. Resin-fixed lipases from R. miehei and C. antarctica exhibited profound differences in activity and FA selectively in response to changes in a(w'), relative to free and Celite-bound forms. These findings suggest that FA selectivity for lipid modification is influenced by a(w) and immobilization, but that each lipase has a characteristic response to these factors in a manner that cannot be predicted.     

The 80s loop of the catalytic chain of Escherichia coli aspartate transcarbamoylase is critical for catalysis and homotropic cooperativity.

The X-ray structure of the Escherichia coli aspartate transcarbamoylase with the bisubstrate analog phosphonacetyl-L-aspartate (PALA) bound shows that PALA interacts with Lys84 from an adjacent catalytic chain. To probe the function of Lys84, site-specific mutagenesis was used to convert Lys84 to alanine, threonine, and asparagine. The K84N and K84T enzymes exhibited 0.08 and 0.29% of the activity of the wild-type enzyme, respectively. However, the K84A enzyme retained 12% of the activity of the wild-type enzyme. For each of these enzymes, the affinity for aspartate was reduced 5- to 10-fold, and the affinity for carbamoyl phosphate was reduced 10- to 30-fold. The enzymes K84N and K84T exhibited no appreciable cooperativity, whereas the K84A enzyme exhibited a Hill coefficient of 1.8. The residual cooperativity and enhanced activity of the K84A enzyme suggest that in this enzyme another mechanism functions to restore catalytic activity. Modeling studies as well as molecular dynamics simulations suggest that in the case of only the K84A enzyme, the lysine residue at position 83 can reorient into the active site and complement for the loss of Lys84. This hypothesis was tested by the creation and analysis of the K83A enzyme and a double mutant enzyme (DM) that has both Lys83 and Lys84 replaced by alanine. The DM enzyme has no cooperativity and exhibited 0.18% of wild-type activity, while the K83A enzyme exhibited 61% of wild-type activity. These data suggest that Lys84 is not only catalytically important, but is also essential for binding both substrates and creation of the high-activity, high-affinity active site. Since low-angle X-ray scattering demonstrated that the mutant enzymes can be converted to the R-structural state, the loss of cooperativity must be related to the inability of these mutant enzymes to form the high-activity, high-affinity active site characteristic of the R-functional state of the enzyme.     

Substrate selectivity of lipases in the esterification of cis/trans-isomers and positional isomers of conjugated linoleic acid (CLA)

The substrate selectivity of several microbial lipases has been examined in the esterification of the conjugated linoleic acid (CLA) isomers cis-9,trans-11-, cis-9,cis-11-, trans-9,trans-11- and trans-10,cis-12-octadecadienoic acid with n-butanol in n-hexane. Lipases from Candida cylindracea and Mucor miehei had a preference for the cis-9,trans-11-octadecadienoic acid, while Chirazyme L-5, a Candida antarctica lipase A, accepted the trans-9,trans-11-fatty acid with a high selectivity. Moreover, lipase from Candida cylindracea and Chirazyme L-5 catalysed the esterification of the cis-9,trans-11-octadecadienoic acid with n-butanol faster than the corresponding reaction of the trans-10,cis-12-fatty acid.     

An alternate sucrose binding mode in the E203Q Arabidopsis invertase mutant: an X-ray crystallography and docking study

In the present study, we report on the X-ray crystallographic structure of a GH32 invertase mutant, (i.e., the Arabidopsis thaliana cell-wall invertase 1-E203Q, AtcwINV1-mutant) in complex with sucrose. This structure was solved to reveal the features of sugar binding in the catalytic pocket. However, as demonstrated by the X-ray structure the sugar binding and the catalytic pocket arrangement is significantly altered as compared with what was expected based on previous X-ray structures on GH-J clan enzymes. We performed a series of docking and molecular dynamics simulations on various derivatives of AtcwINV1 to reveal the reasons behind this modified sugar binding. Our results demonstrate that the E203Q mutation introduced into the catalytic pocket triggers conformational changes that alter the wild type substrate binding. In addition, this study also reveals the putative productive sucrose binding modus in the wild type enzyme.     

Characterization of differences in substrate specificity among CYP1A1, CYP1A2 and CYP1B1: an integrated approach employing molecular docking and molecular dynamics simulations

Recent trends in new drug discovery of anticancer drugs have made oncologists more aware of the fact that the new drug discovery must target the developing mechanism of tumorigenesis to improve the therapeutic efficacy of antineoplastic drugs. The drugs designed are expected to have high affinity towards the novel targets selectively. Current research highlights overexpression of CYP450s, particularly cytochrome P450 1A1 (CYP1A1), in tumour cells, representing a novel target for anticancer therapy. However, the CYP1 family is identified as posing significant problems in selectivity of anticancer molecules towards CYP1A1. Three members have been identified in the human CYP1 family: CYP1A1, CYP1A2 and CYP1B1. Although sequences of the three isoform have high sequence identity, they have distinct substrate specificities. The understanding of macromolecular features that govern substrate specificity is required to understand the interplay between the protein function and dynamics, design novel antitumour compounds that could be specifically metabolized by only CYP1A1 to mediate their antitumour activity and elucidate the reasons for differences in substrate specificity profile among the three proteins. In the present study, we employed a combination of computational methodologies: molecular docking and molecular dynamics simulations. We utilized eight substrates for elucidating the difference in substrate specificity of the three isoforms. Lastly, we conclude that the substrate specificity of a particular substrate depends upon the type of the active site residues, the dynamic motions in the protein structure upon ligand binding and the physico‐chemical characteristics of a particular ligand. Copyright © 2016 John Wiley & Sons, Ltd.     

Structural and dynamic roles of permanent water molecules in ligand molecular recognition by chicken liver bile acid binding protein

Chicken liver bile acid binding protein (cL-BABP) crystallizes with water molecules in its binding site. To obtain insights on the role of internal water, we performed two 100 ns molecular dynamics (MD) simulations in explicit solvent for cL-BABP, as apo form and as a complex with two molecules of cholic acid, and analyzed in detail the dynamics properties of all water molecules. The diffusion coefficients of the more persistent internal water molecules are significantly different from the bulk, but similar between the two protein forms. A different number of molecules and a different organization are observed for apo- and holo-cL-BABP. Most water molecules identified in the binding site of the apo-crystal diffuse to the bulk during the simulation. In contrast, almost all the internal waters of the holo-crystal maintain the same interactions with internal sidechains and ligands, which suggests they have a relevant role in protein-ligand molecular recognition. Only in the presence of these water molecules we were able to reproduce, by a classical molecular docking approach, the structure of the complex cL-BABP::cholic acid with a low ligand root mean square deviation (RMSD) with respect to its reference positioning. Literature data reported a conserved pattern of hydrogen bonds between a single water molecule and three amino acid residues of the binding site in a series of crystallized FABP. In cL-BABP, the interactions between this conserved water molecule and the three residues are present in the crystal of both apo- and holo-cL-BABP but are lost immediately after the start of molecular dynamics. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Endoxylanase substrate selectivity determines degradation of wheat water-extractable and water-unextractable arabinoxylan

The relative activity of an endoxylanase towards water-unextractable (WU-AX) and water-extractable arabinoxylan (WE-AX) substrates, referred to as endoxylanase substrate selectivity, impacts the enzyme functionality in cereal-based biotechnological processes such as bread-making and gluten starch separation. A set of six endoxylanases representing a range of substrate selectivities as determined by a screening method using chromophoric substrates [Anal. Biochem.2003, 319, 73-77] was used to examine the impact of such selectivity on changes in structural characteristics of wheat WU-AX and WE-AX upon enzymic hydrolysis. While WE-AX degradation by the selected endoxylanases was very comparable with respect to apparent molecular mass (MM) profiles and arabinose to xylose ratio of the hydrolysates formed, WU-AX solubilisation and subsequent degradation of solubilised fragments gave rise to widely varying MM profiles, depending on the substrate selectivity of the enzymes. Enzymes with high selectivity towards WU-AX de facto generated higher MM fragments from WU-AX than enzymes with low selectivity. The arabinose to xylose ratios of solubilised fragments were independent of the degree of solubilisation.