Surface Binding Sites (SBSs), Mechanism and Regulation of Enzymes Degrading Amylopectin and α-Limit Dextrins

Abstract

Certain enzymes interact with polysaccharides at surface binding sites (SBSs) situated outside of their active sites. SBSs are not easily identified and their function has been discerned in relatively few cases. Starch degradation is a concerted action involving GH13 hydrolases. New insight into barley seed α-amylase 1 (AMY1) and limit dextrinase (LD) includes i) kinetics of bi-exponential amylopectin hydrolysis by AMY1, one reaction having low K_m (8 µg/mL) and high k_cat (57 s^－1) and the other high K_m (97 µg/mL) and low k_cat (23 s^－1). β-Cyclodextrin (β-CD) inhibits the first reaction by binding to an SBS (SBS2) on domain C with K_d = 70 µM, which for the SBS2 Y380A mutant increases to 1.4 mM. SBS2 thus has a role in the fast, high-affinity component of amylopectin degradation. ii) The N-terminal domain of LD, the debranching enzyme in germinating seeds, shows distant structural similarity with domains including CBM21 present in other proteins and involved in various molecular interactions, but no binding site identity. LD is controlled by barley limit dextrinase inhibitor (LDI) which belongs to the cereal-type inhibitor family and forms a tight 1:1 complex with LD. iii) LDI in turn is regulated by disulfide reduction mediated by the barley thioredoxin h (trxh) NADPH-dependent thioredoxin reductase (NTR) system. Based on the progress monitored by released free thiol groups from LDI and its failure to inhibit LD as elicited by trxh, the LDI inactivation is proposed to stem from loss of structural integrity due to reduction of all four disulfides.

INTRODUCTION

Although carbohydrate surface binding sites (SBSs) were first described more than three decades ago in glycogen phosphorylase and later in α-glucan phosphorylases from plants,^{1) 2) 3)} functional roles of SBSs (sometimes referred to as secondary binding sites) situated outside of the active site area in polysaccharide metabolizing enzymes for many years received only sporadic attention. Increasing interest in SBSs, however, emerged during the past few years along with identification of several SBSs as well as further assignment of their functional properties. The slow progress with learning more about SBSs probably reflects that they have not been considered essential for the enzyme catalytic activities combined with technical difficulties at several levels, i.e., firstly with the actual identification of SBSs, and secondly in characterizing their mechanism of action and impact on the enzyme function and activity. While SBSs originally were observed in both starch-related α-glucan^{3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17) 18)} and glycogen^{1) 2)} converting enzymes, the later disclosed presence of SBSs in certain cell wall polysaccharide degrading enzymes of potential application in biomass utilization,^{19) 20) 21) 22)} indicates SBSs to be a more widely occurring generic concept in polysaccharide metabolizing enzymes.

Two SBSs (SBS1 and SBS2) in barley α-amylase were first identified by differential chemical modification²³⁾ and crystallography^{6) 8) 24)} and previously demonstrated by aid of site-directed mutagenesis in barley α-amylase isozyme 1 (AMY1) to be important for activity on starch granules and polysaccharide substrates.^{4) 9) 10) 11) 25)} Most recently, further distinction between the individual functional roles of SBS1 and SBS2 was achieved through detailed analysis of the effect of SBS2 in amylopectin catalyzed hydrolysis by AMY1 showing a non-Michaelis-Menten kinetic behaviour.²⁶⁾

Regulation of hydrolases acting on starch and other poly- and oligosaccharides can also happen through interaction with proteinaceous inhibitors^{27) 28) 29) 30) 31) 32) 33) 34)} as well as by ligands binding at allosteric sites^{15) 35) 36) 37)} or by post-translational modification.³⁸⁾ Structural insights into complex formation between polysaccharide hydrolyzing enzymes and their specific proteinaceous inhibitors were first gained on α-amylases,^{27) 28) 29) 30) 31)} followed by xylanases^{31) 32) 33)} and pectin methyl esterases³⁴⁾ and furthermore includes characterization of a related glycosidase/invertase inhibitor.³⁹⁾ Very recently, we solved the crystal structure of a unique complex of a debranching enzyme and the corresponding inhibitor, i.e. barley limit dextrinase (LD)^{40) 41) 42)} and its endogenous inhibitor (LDI)^{43) 44) 45) 46) 47)} that belongs to the cereal-type inhibitor protein family.^{29) 30) 48) 49) 50)} Most remarkably, the LD:LDI complex (Møller et al., unpublished) is distinct from the structure already determined for barley α-amylase isozyme 2 (AMY2) in complex with barley α-amylase/subtilisin inhibitor (BASI)²⁸⁾ while noticeably both BASI and LDI are targets of the cytosolic h-type thioredoxin (trxh) present in mature barley seeds (HvTrxh).^{51) 52) 53) 54)} This latter redox-related control by trxh confers an additional level of regulation that similarly to the enzyme:inhibitor complex formation involves protein-protein interaction. By contrast to the very clear preference of HvTrxh for one of the two disulfide bonds found in BASI,^{51) 52)} apparently all four disulfides of LDI as well as the Cys59-glutathione disulfide bond are susceptible to reduction by HvTrxh1 and HvTrxh2,⁵⁴⁾ as will be further discussed below.

Establishing a toolbox for identification and characterization of carbohydrate surface binding sites in polysaccharide processing enzymes. The SBSs are different from carbohydrate binding modules (CBMs), but in many respects SBSs seem to exert the same functions as CBMs. An obvious short-coming for understanding enzyme-polysaccharide interactions is the lack of structural insight at the atomic resolution level into the binding of macromolecular ligands to the enzyme surface. This situation encourages the development of a dedicated toolbox comprising procedures for identifying as well as characterizing SBSs and their mode of action. In a recent review, Cuyvers et al.⁵⁵⁾ cover the occurrence of SBSs in polysaccharide processing enzymes based on published crystal structures of carbohydrate-active enzymes having oligosaccharides accommodated at putative SBSs. These authors list seven possible functional roles of SBSs; i) targeting the enzyme to its substrate; ii) guiding substrate chains into the enzyme’s catalytic site; iii) disruption of substrate interactions to facilitate hydrolysis of its single polysaccharide chains; iv) increasing the frequency of multiple attacks; v) introduction of sites for allosteric regulation; vi) delivery of reaction products, e.g., to another enzyme or a membrane transport unit and vii) anchoring of the enzyme to the cell wall of the parent microorganism. A couple of additional roles of SBSs can be included; viii) bias enzyme specificity by giving preference for a particular substrate reaction, as will be expanded below in relation to amylopectin hydrolysis catalyzed by AMY1;²⁶⁾ and ix) acting as binding sites for pharmaceutical molecular chaperones as demonstrated for GH27 human α-galactosidase, where an SBS interacting with β-galactose is proposed to advance folding of the recombinant therapeutic enzyme.⁵⁶⁾

A number of techniques facilitate recognition of SBSs. Crystallography is particularly informative as it can disclose carbohydrate ligands bound on the enzyme surface at a certain distance from the active site.^{12) 13) 14) 15) 16) 17) 18) 20) 21) 22)} While most cases of putative SBSs have been discovered by protein crystallography,^{2) 5) 6) 7) 8) 12) 13) 14) 15) 16)17) 18) 19) 20) 21) 22)} at least one example was reported for the structure of a xylanase in complex with xylooligosaccharides solved by NMR.⁵⁷⁾

Amino acid residues proposed to play a role in putative SBSs can be further investigated by site-directed mutagenesis followed by various assays validating their properties, e.g., surface plasmon resonance (SPR), which is both sensitive and flexible in accurately analyzing oligosaccharide binding even when the affinity is rather modest and which may also provide useful insight into interaction with polysaccharide.^{4) 9) 10) 11) 25) 58) 59) 60) 61)} Another simple albeit less exact characterization technique is affinity electrophoresis (AE), which has been used early on to determine binding constants of glycogen phosphorylases to glycogen and to validate functional roles of SBSs using engineered chimeric forms of plant phosphorylases.^{1) 3) 62) 63)} In AE, the migration rate of the investigated enzyme in native PAGE gels cast to contain the interacting polysaccharide candidate is compared with the migration rate of the enzyme in a gel run in parallel but without the potential polysaccharide substrate. While about 10% of starch-metabolizing enzymes contain one or more starch binding domains (SBDs) that provide contact with starch granules and related polymeric substrates and which based on sequence similarity are organized in 10 CBM families in the CAZy database (http://www.cazy.org/),⁶⁴⁾ there is no good estimate of the number of starch-active enzymes possessing SBSs. Barley AMY1 has no SBD, but contains two SBSs and its migration is retarded in AE performed by native PAGE with amylopectin included in the gel (Fig. 1). Insoluble polysaccharide “pull-down” is another SBS screening technique and it is anticipated that also carbohydrate microarrays will be useful for identification of the presence of potential SBSs.

Fig. 1

Affinity electrophoresis analysis of the interaction between the barley α-amylase isoform 1 (AMY1) and potato amylopectin.

On the left is a control native PAGE gel and on the right is an identical gel except that 0.1% amylopectin is included. The outside lanes consist of a protein marker (NativeMark^TM from Invitrogen) and the inner lanes are AMY1. The gels are 12% acrylamide and the buffer system is Tris-borate pH 8.7. The proteins were separated at 55 V for 17 h at 4°C.

Obviously, a weakness in most SBS screening techniques is the interference of the carbohydrate ligand with the active site. For both SPR and AE, this interaction may be prevented by blocking the active site through reaction with a mechanism-based inhibitor that gets covalently bound to a catalytic residue and blocks substrate binding to the active site. Such an approach has enabled discrimination of binding to SBSs and at the active site of xylanase A from Bacillus subtilis.⁵⁸⁾ While this type of strategy may in principle work for any carbohydrate-active enzymes, the methods and availability of inhibitors for blocking active sites are enzyme-dependent. In fact these reagents are unavailable for the majority of carbohydrate-active enzymes and require synthesis of suitable, functionalized carbohydrate derivatives. Alternatively, if an enzyme inhibitory carbohydrate derivative is accessible which has significantly higher affinity for the active site than for the SBSs, this may offer a way to selectively block the active site. However, the discrimination between active sites and SBSs is tricky and acarbose, a powerful inhibitory pseudotetrasaccharide for several amylolytic enzymes, has also been observed to bind at SBSs.^{6) 7) 18) 24) 65)}

SBS1 and SBS2 in barley α-amylase 1 (AMY1) and the role of SBS2 in hydrolysis of amylopectin and amylose. SBS1 and SBS2 in AMY1, previously named “the starch granule-binding surface site” and “the pair of sugar tongs”, respectively,^{6) 8) 9) 10) 11) 24) 25)} both interact with maltoheptaose, other maltooligosaccharides and acarbose as observed in crystal structures of different complexes with wild-type and the inactive catalytic nucleophile D180A AMY1 mutant, respectively (Fig. 2).^{8) 24)} A series of single, double and triple SBS mutants involving replacement of aromatic residues stacking onto and/or hydrogen bonding with carbohydrate ligands at SBS1 and SBS2 in the inactive D180A and wild-type AMY1,^{4) 10) 11) 24)} previously allowed us to propose functional roles of SBS1 and SBS2, notably including effects on the degree of multiple attack on amylose⁶⁶⁾ and also on the subsite map.¹¹⁾ SBS1 is a carbohydrate binding site where Trp278 and Trp279 (Trp277 and Trp278 in AMY2⁶⁾) stack onto two adjacent glucosyl units of maltooligosaccharides and acarbose in crystal structures of AMY1 and AMY2.^{6) 8) 24)} SBS2 of AMY1 revealed a 3.1 Å movement of the side chain of the central residue Tyr380 upon carbohydrate binding. Tyr380 contributes with a large number of ligand contacts. ^{8) 24)} Single W278A and W279A AMY1 mutants at SBS1 caused greater loss of starch granule affinity and hydrolysis than in case of Y380A at SBS2 and the affinity of the double W278A/W279A mutant was further reduced (Fig. 3).^{9) 11)} The SBS1 and SBS2 triple mutant W278A/W279A/Y380A showed no detectable binding to starch granules and the rate of release of soluble reducing hydrolysis products decreased to about 0.25% of that of AMY1 wild-type.¹¹⁾ The two SBSs thus possess differential affinity to starch granules and they synergistically advance mobilization of starch. Single α-glucan chains in amorphous regions on the starch granules may bind to SBS2. Furthermore recent data propose that SBS2 has a specific role in the kinetics of amylopectin hydrolysis.^{11) 25) 26)} GH13 is the largest glycoside hydrolase family⁶⁴⁾ containing more than 30 subfamilies⁶⁷⁾ and the SBS2 in AMY1^{8) 9) 10) 11) 24) 25) 66)} and AMY2²⁵⁾ represents a remarkable case of a very important function residing in a C-terminal domain of a GH13 member.

Fig. 2

Crystal structure of the inactive catalytic nucleophile mutant Asp180Ala of barley AMY1 in complex with maltoheptaose (PDB entry 1rp8).

In red is a cartoon of the secondary structure of the enzyme, with a transparent molecular surface. The maltoheptaose is shown as grey sticks and the SBS1 and SBS2 are shown as thickened sticks and indicated by blue arrows. The active site is indicated by a red arrow.

Fig. 3

Confocal laser scanning microscopy of the interaction between barley starch granules and Fluorescein-5-EX-labeled AMY1 wt and mutants.

The dissociation constants were determined using Langmuir binding analysis towards barley starch granules.^{9) 11)} For the combined SBS1 + SBS2 mutants (W278A/Y380A and W279A/Y380A) no binding to the starch granule surface was visible (data not shown), which correlates well with that no starch binding was measured using Langmuir binding analysis.^{9) 11)}

Wild-type and Y380A AMY1 hydrolyzed amylose with k_cat and K_m of 132 s^－1 and 0.22 mg/mL and 108 s^－1 and 0.37 mg/mL, respectively, indicating that Y380A is not seriously affecting the steady-state kinetics of the hydrolysis of the linear substrate.^{9) 24)} In contrast to conventional Michaelis-Menten kinetics of product formation from amylose, however, the initial rates of hydrolysis of amylopectin could not be determined, since the K_m was too low to allow colorimetric quantification of reducing maltooligosaccharide products, of which most had a degree of polymerization (DP) of 7.²⁶⁾ Entire progress curves were therefore monitored to determine kinetic parameters for amylopectin hydrolysis. While the progress curve for degradation of amylose followed a simple mono-exponential function, the hydrolysis of amylopectin appeared to follow a bi-exponential function that described the kinetics of two reactions, “a” and “b”, for the degradation corresponding to fractions about 0.40 and 0.60 of the oligosaccharide products.²⁶⁾ These two reactions for catalysis of amylopectin hydrolysis by wild-type AMY1, were characterized by k_cat and K_m of 57 s^－1 and 8 µg/mL (for “a”) and 23 s^－1 and 97 µg/mL (for “b”) (Fig. 4). ²⁶⁾ The AMY1 Y380A mutant essentially retained these k_cat values, whereas the K_m of the fast reaction (“a”) increased by a factor of 20, while K_m was only slightly increased for the slow reaction (“b”) compared to wild-type AMY1. A similar effect was obtained by adding 5 mM β-cyclodextrin (β-CD) to the amylopectin hydrolysis catalyzed by wild-type AMY1. K_d of β-CD binding by wild-type AMY1 is about 10 times lower than the corresponding K_d for Y380A AMY1 as measured by SPR.^{9) 10) 11) 25)} Noticeably, in agreement with the kinetics of the hydrolysis of amylopectin by the Y380A mutant, AMY1 was not critically affected by the presence of 5 mM β-CD.²⁶⁾ It is concluded that SBS2 provides a functional advantage to AMY1 by specific binding to amylopectin clusters, which leads to saturation of the active site at a lower substrate concentration. AMY1 thus seems to belong to a small group of α-amylases containing a functional SBS2, which might explain the dramatic difference in the apparent K_m of this enzyme and that of α-amylase from Aspergillus oryzae.⁶⁸⁾

Fig. 4

Saturation of the amylopectin degradation reaction catalyzed by AMY1.

Initial rates, represented by v, calculated from progress curves are shown for the combined reaction (closed squares), the “a” reaction (open squares) and the “b” reaction (open diamonds). The kinetic parameters (K_m; k_cat) calculated using the Michaelis-Menten equation corresponding to the curves shown were: (closed squares) K_m 23 µg/mL; k_cat 80 s^－1, (open squares) K_m 8 µg/mL; k_cat 57 s^－1, (open diamonds) K_m 97 µg/mL determined using a fixed value for k_cat of 23 s^－1 calculated by subtracting k_cat for the “a” reaction from that of the combined reaction. Reprinted from Ref. 26) with permission from Elsevier.

Regulatory processes in germinating barley seeds involving proteinaceous enzyme inhibitors and redox reactions. Numerous interplaying processes in the germinating barley seed result in an optimal utilization of storage compounds and ensure the successful development of the plantlet. Regulation of germination takes place at several levels. At the gene level mediated by phytohormones, e.g., gibberellic acid that is synthesized by the embryo after water uptake at the onset of germination and diffused to the aleurone layer. In addition, several enzyme activities involved in germination are regulated by processes that involve protein-protein interactions. In the case of starch mobilization, these include the straightforward inhibition of hydrolases of starch and limit dextrins, i.e., barley α-amylase 2 (AMY2) by barley α-amylase/subtilisin inhibitor (BASI)²⁷⁾²⁸⁾ and limit dextrinase (LD) by limit dextrinase inhibitor (LDI).⁴³⁾⁴⁶⁾ Moreover, regulation of the enzyme activities by the endogenous proteinaceous inhibitors BASI and LDI appears to occur at an additional level consisting in disulfide bond reduction in the inhibitory proteins as catalyzed by the thioredoxin system composed of thioredoxin type h (two isoforms are found in barley)⁶⁹⁾ and NADPH-dependent thioredoxin-reductase (NTR; also two isoforms in barley).⁷⁰⁾ During the past decade we identified and characterized the key proteins in the barley thioredoxin system (HvTrxh1, HvTrxh2, HvNTR1 and HvNTR2) as well as a large number of trxh potential target proteins, including the identification of individual specific target disulfide bonds in these proteins.^51)71)72) Noticeably, the structure was solved of the disulfide-bonded trapped complex between HvTrxh2 and barley α-amylase inhibitor (BASI)⁵²⁾ that involves one of the two disulfide bonds present in BASI. This trapped complex of HvTrxh2 and BASI was the first crystal structure providing detailed insight into the elements that determine recognition between a thioredoxin and its target protein. Very recently, the reaction of HvTrxh1 and HvTrxh2 with LDI was investigated in solution⁵⁴⁾ as will be further described below.

Three-dimensional structure of native barley limit dextrinase (LD). A large group of enzymes belonging to GH13 specifically catalyze hydrolysis of endo-α-1,6 linkages such as branch points in amylopectin and/or related poly- and oligosaccharides,⁶⁴⁾⁶⁷⁾ e.g., α- and β-limit dextrins as well as of α-1,6 linkages in the linear fungal exopolysaccharide pullulan from Aureobasidium pullulans.^42)73)74) Limit dextrinase (LD) is the sole enzyme that debranches α-limit dextrins in the germinating barley seed. It also shows high activity towards pullulan,^40)41)42) but has much lower activity towards amylopectin itself.⁷⁵⁾ LD plays an important role in starch mobilization and is perhaps participating in starch biosynthesis by aiding with restructuring the organization and clustering of branches in intermediate structures of amylopectin sometimes referred to as phytoglycogen.⁷⁶⁾

LD was produced recombinantly in high yields by secretory expression using Pichia pastoris as host and applying a high cell-density fermentation procedure.⁴²⁾ The quality of the recombinant LD was excellent as its activity (k_cat,app/K_m,app = 488 ± 23 mL･mg^－1･s^－1) was 3.5 fold higher than of LD previously purified from germinating barley seeds.⁴²⁾ Kinetic parameters for hydrolysis of pullulan were K_m,app = 0.16 ± 0.02 mg･mL^－1 and k_cat,app = 79 ± 10 s^－1 as fitted to a Michaelis-Menten equation including a substrate inhibition term.⁴²⁾ This distinct kinetic behavior may reflect rather than a direct inhibition by substrate that LD has a high transglycosylation activity, as supported by preliminary experiments, e.g., including LD catalyzed maltosylation of α-CD (Vester-Christensen et al., unpublished).

The availability of the recombinant protein made it possible to determine the three-dimensional structure of LD, initially in complex with α- and β-CDs (PDB entries 2y5e and 2y4s), which are inhibitors with K_d of 27 and 0.7 µM, respectively.⁴²⁾ LD is a multidomain enzyme of subfamily GH13_13⁶⁷⁾ composed of an N-terminal domain, followed by a CBM48 connected to the catalytic (β/α)₈-barrel domain followed by a C-terminal β-sandwich domain of two sheets each with four β-strands flanked by four short α-helices.⁷⁷⁾ In the LD α- and β-CD complexes, the structure of LD was not defined for three short loops in the N-terminal domain. Recently, however, a crystal structure was obtained of free LD (PDB entry 4aio) in which these loops were defined (Fig. 5).⁷⁸⁾ Catalytic and key ligand binding residues at the active site superimposed very well between the free and the β-CD complex LD structures (PDB entry 2y4s).⁷⁸⁾ Compared, however, with pullulanase from Klebsiella pneumonia that belongs to the same subfamily (GH13_13) in free state and with ligand bound at the active site, two different main chain conformations were seen in the loop EGWDS containing the catalytic acid/base Glu706.⁷³⁾⁷⁸⁾ Moreover, Trp708 made a～90° rotation to stack at the subsite + 2 of the active site with bound maltose, maltotriose or maltotetraose (PDB entries 2fhb, 2fhc or 2fhf, respectively) relative to the rotamer of Trp708 in native pullulanase (PDB entry 2fgz) or in complexes with glucose (PDB entry 2fh6) or isomaltose (PDB entry 2fh8), which was seen to adopt the “inactive” rotamer conformation.⁷³⁾ Noticeably, the conserved barley LD sequence motif EGWDF that contains the catalytic acid/base protein donor Glu510, in structures of both free LD and complexes with α- or β-CD LD adopts an “active” conformation and LD Trp512 (Fig. 5) was also found as the “active” rotamer.⁷⁷⁾⁷⁸⁾ This apparently fixed conformation of the central element from the LD active site proposes that the active site has less flexibility in LD as compared to the pullulanase, possibly explaining the low activity of LD towards amylopectin and the high activity towards limit dextrins as compared to bacterial pullulanases.⁷⁷⁾⁷⁸⁾

Fig. 5

Overall structure of LD (PDB entry 4aio).

N-domain, green; CBM48, red; catalytic domain, gray; C-domain, blue; Ca²⁺, purple. The catalytic site residues (Asp473, Glu510 and Asp642) are shown as orange sticks, while Trp512 is shown as gray sticks. The three short loops, which were unsolved in the structures of LD in complex with the competitive inhibitors α- or β-CD (PDB entries 2y5e and 2y4s), are indicated by arrows.

The unresolved issue concerning the possible role of the LD N-terminal domain motivated applying several alignment methods against the entire PDB archive, which led to identification of nine unique structures.⁷⁸⁾ Five of these were domains found in various pullulanases of GH13_13 and GH13_14, but the sequence identity to LD was generally low (7–13%) except for the domain from K. pneumonia pullulanase which showed 31% sequence identity with the N-terminal domain of LD. Three other domains have documented albeit diverse functions including binding of a peptide ligand, domain multimerization and N-acetyl-D-glucosamine binding (Fig. 6).⁷⁸⁾ The ligand binding sites in these domains, however, do not overlap. Noticeably the starch binding domain of CBM21 from Rhizopus oryzae glucoamylase showed 6% sequence identity and was identified with slightly lower score as the structurally similar domains mentioned above (Fig. 7).⁷⁸⁾⁷⁹⁾ Only three shared residues were surface exposed among the identical residues from the structure-based sequence alignment to the CBM21 of the N-terminal domain of LD, and these residues were located in a part of CBM21 that was not structurally conserved. Furthermore, the identified starch binding residues in the CBM21 were neither conserved in the N-terminal domain of LD nor replaced by residues of similar biophysical properties.⁷⁸⁾⁷⁹⁾ Therefore, the N-terminal domain of LD may participate in molecular interactions, but there is no evidence that suggests whether this is with substrate, other proteins, or in multimerization.⁷⁸⁾

Fig. 6

Superimposition of the N-domain of LD (orange) with structurally similar domains with known function: (a) Erythropoietin receptor (PDB entry 1eba; red) and the two ligands erythropoietin-mimic peptide, EMP33, and 3,5-dibromotyrosine, DBY-T), (b) Cytokine receptor γ-chain (PDB entry 2b5i; blue) N-acetyl-D-glucosamine (NAG), (c) Esterase (PDB entry 3doi; green) and diethyl phosphonate (DEP). Reprinted from Ref. 78).

Fig. 7

Structural alignment of the N-terminal domain of LD (PDB entry 4aio,⁷⁸⁾ green) and CBM21 from Rhizopus oryzae (PDB entry 2djm,⁷⁹⁾ blue) including a β-CD molecule from a later structure of the CBM21 from Rhizopus oryzae (PDB entry 2v8l, purple sticks) superimposed onto the binding site.

The activity of LD which is implicated both in the biosynthesis and degradation of starch in vivo⁷⁶⁾ is regulated by the endogenous LDI⁴³⁾ that belongs to the cereal-type inhibitor protein family containing numerous inhibitors of α-amylases and other hydrolases, typically the proteases trypsin or chymotrypsin.²⁷⁾ These inhibitors are also referred to as CM-proteins, which are abundant in barley and other cereal seeds. The LDI was recently produced recombinantly in high yields using P. pastoris as host.⁴⁷⁾ It has a molecular weight of about 13 kDa and contains four intramolecular disulfide bonds and one glutathionylated cysteinyl residue, which in the LDI purified from barley seeds was found both in glutathionylated and cysteinylated forms.^44)47)54) LD:LDI forms a 1:1 molar complex⁴⁵⁾⁴⁷⁾ (Fig. 8) of very high affinity.⁴⁶⁾ In barley, the LD:LDI complex has a counterpart in AMY2: BASI.^27)28)80)81) Both of these inhibitors may undergo simultaneous regulation since they share the sensitivity to the disulfide reductase trxh.⁵¹⁾⁵²⁾ It appears, however, that the mode of action of HvTrxh on these two inhibitors is distinctly different. Furthermore, LDI possessed a remarkably high thermal stability of importance for the beer production.⁴⁷⁾

Fig. 8

The relative inhibition (%) of LD activity at different LDI molar ratios assayed by using soluble pullulan as substrate.

The assay was done at 37°C and pH 5.5.⁴⁷⁾ The open circles represent the means of a triplicate data set and the error bars depict standard deviations. The solid line is a smooth fit to the data to aid clarity. Reprinted from Ref. 47) with permission from Elsevier.

The interaction between barley thioredoxin h (trxh) and limit dextrinase inhibitor (LDI). The LDI is a target for barley thioredoxin.⁵³⁾ However, binding of LDI to LD prevented HvTrxh1 from inactivating LDI.⁵⁴⁾ In the case of two cereal-type inhibitors related to LDI (BMA1-1 and BMA1-2).⁵¹⁾ as well as of BASI.⁵²⁾ HvTrxh1 reduced a specific disulfide bond. This was apparently not the case for LDI. By contrast the loss of LDI inhibitory activity accumulated with increasing release of free thiol groups by treatment with HvTrxh1 or HvTrxh2 (Fig. 9).⁵⁴⁾ Noticeably, the glutathione disulfide-linked to Cys59 was reduced before complete release of 10 thiol groups per LDI molecule. The reduction of the Cys59-glutathione disulfide seemed not accompanied by loss of LDI activity.⁵⁴⁾

Fig. 9

LDI reduction by HvTrxh1 (○) and HvTrxh2 (□) monitored as release of thiol groups (――), and loss of LDI activity (……).

Insert shows release of thiol groups (0–60 min). Conditions: 20 µM LDI, 4 µM HvTrxh1 or HvTrxh2, 0.1 µM HvNTR2, 4 mM NADPH in 100 mM Tris-HCl, pH 8.0 (RT). Reprinted from Ref. 54) with permission from Elsevier.

CONCLUDING REMARKS

There is increasing acceptance that SBSs contribute to characteristic functional properties of polysaccharide converting enzymes, including amylolytic enzymes, in their interplay with polymeric and supramolecular carbohydrate structures such as starch granules and plant cell walls. The lack of high-resolution structures illustrating enzyme complexes with polysaccharides is compensated by mutational analysis including assay of both carbohydrate binding and hydrolysis to gain knowledge of the mechanism of action and importance of SBSs in various multivalent substrate binding events. The assignment of a specific SBS in AMY1 (SBS2) for a role in the fast, high affinity-part of amylopectin degradation is one example of SBSs constituting a field open for discoveries of fundamental properties. Such new understanding of SBSs may be tested by introduction of SBSs using rational protein engineering strategies to confer manipulated or new functionalities and possesses strong potential for advancing industrial-scale conversions of biomass and also of starch and related sugars.

The progress in analysis of structure/function relationships of LDI and the interaction with LD fundamentally expands insight into the mode of action of proteinaceous inhibitors with starch-degrading enzymes; in particular it reveals an unexpected diversity of target enzyme recognition motifs in members of the cereal inhibitor family. The high thermal stability of LDI⁴⁷⁾ and its fold-similarity with related proteinaceous inhibitors suggest that the LDI fold can provide a starting point for engineering recognition and diverse specificities to be confer a robust molecule with desired requirements.

ACKNOWLEDGMENTS

We are grateful to our colleagues Andreas Blennow, Camilla Christiansen, Rob Field, Martin Rejzek, Hans Erik Mølager Christensen, Hiroyuki Nakai, Richard Haser and Nushin Aghajari for stimulating discussions. This work was supported by the Danish Natural Science Research Council, the Danish Research Council for Technology and Production Sciences, the Carlsberg Foundation, an H.C. Ørsted postdoctoral fellowship from the Technical University of Denmark (to DC), Ph. D. fellowships from the Technical University of Denmark (to MSM, JMA, MBVC, MMN, CW), Danish National Advanced Technology Foundation (HTF), Maersk Oil and Gas A/S and the University of Copenhagen (all to MW and JWN) and an Oticon Foundation Master thesis stipend (to JMJ).

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