Crystal Structure of Amylomaltase from Corynebacterium glutamicum

Article pubs.acs.org/JAFC

Crystal Structure of Amylomaltase from Corynebacterium glutamicum Seongjoon Joo,†,‡ Sangwoo Kim,†,‡,§ Hogyun Seo,† and Kyung-Jin Kim*,† †

Structural and Molecular Biology Laboratory, School of Life Sciences and Biotechnology, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea § School of Nano-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 689-798, Republic of Korea ABSTRACT: Amylomaltase is an essential enzyme in maltose utilization and maltodextrin metabolism, and it has been industrially used for the production of cyclodextrin and modification of starch. We determined the crystal structure of amylomaltase from Corynebacterium glutamicum (CgAM) at a resolution of 1.7 Å. Although CgAM forms a dimer without NaCl, it exists as a monomer in physiological concentration of NaCl. CgAM is composed of N- and C-terminal domains, which can be further divided into two and four subdomains, respectively. It exhibits a unique structural feature at the functionally unknown Ndomain and also shows two striking differences at the C-domain compared to other amylomaltases. These differences at extended edge of the substrate-binding site might affect substrate specificity for large cyclodextrin formation. The bis-tris methane and sulfate molecules bound at the substrate-binding site of our current structure mimic the binding of the hydroxyl groups of glucose bound at subsites −1 and −2, respectively. KEYWORDS: Corynebacterium glutamicum, amylomaltase, maltose, maltodextrin

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INTRODUCTION

polysaccharide-forming enzyme that catalyzes a reversible transglycosidase reaction.13,16 It utilizes variable-length maltodextrins as donors and acceptors to transfer α-glucosyl, maltosyl, and longer dextrinyl units, which results in disproportionation. Therefore, amylomaltase forms variablelength maltodextrins and glucose. However, it can also take the nonreducing end of the donor substrate as the acceptor substrate, which results in the formation of a cyclodextrin like cyclodextrin glucanotransferase (CGTase).17−19 The reverse of this cyclization reaction is often referred to as the “coupling reaction”.17 In addition, amylomaltases also have a weak cyclomaltodextrinase activity that is ability to hydrolyze cyclodextrins, whereby ring opening occurs.20,21 The synthesis of cyclodextrin by amylomaltases makes these enzymes attractive biocatalysts for producing pharmaceutically important materials and valuable fine chemicals.22−24 Therefore, many structural studies have been carried out on amylomaltases, revealing their molecular functions. Such studies have often focused on thermophilic bacteria such as Thermus aquaticus, Thermus thermophilus HB8, Thermus brockianus, and Aquifex aeolicus.20,25,26 Amylomaltase from C. glutamicum (CgAM) is a biologically and industrially important enzyme. CgAM was characterized, analyzed, engineered, and crystallized by Pongsawasdi et al. for cyclodextrin production and starch modification, notwithstanding a report on the crystal structure of CgAM.21,27−32 Recently, a crystal structure of amylomaltase (MalQ) from mesophilic E. coli, which has an amino acid similarity of 30% to CgAM, has been reported.33 EcMalQ has a novel N-domain that is also present in CgAM. In this study, we determined the crystal

Corynebacterium glutamicum has attracted the attention of the biotechnology industry and is employed in large-scale fermentation owing to its remarkable ability to produce amino acids, nucleotides, and vitamins.1 The organism utilizes a variety of external mono- and disaccharides, alcohols, and organic acids as single or combined sources of carbon and energy.2 C. glutamicum cells growing in a medium containing sugars accumulate glycogen and degrade that polymer when sugar becomes a limiting factor.3,4 Unlike other Gram-positive bacteria, C. glutamicum has a glycogen degradation pathway similar to that in Escherichia coli.5 During the degradation of glycogen, which is intertwined with maltose metabolism, C. glutamicum forms maltodextrins.4,5 Enzymes such as glycogen phosphorylase (GlgP), glycogen debranching enzyme (GlgX), glucokinase (Glk), α-phosphoglucomutase (α-Pgm), maltodextrin glucosidase (MalZ), maltodextrin phosphorylase (MalP), and 4-α-glucanotransferase (MalQ) participate in maltose/ maltodextrin metabolism and glycogen degradation in E. coli (Figure 1).5,6 In C. glutamicum, maltose and maltodextrins serve as substrates in the synthesis of trehalose. Trehalose is involved in the response to osmotic stress and also in the biosynthesis of mycolic acids, which are an important component of the Corynebacteriaceae cell envelope.7 4-α-Glucanotransferase which catalyzes the glucan transfer reaction is structurally and mechanistically related to αamylases (family 13 of the glycoside hydrolases or GH13).8−10 Amylomaltase (EC 2.4.1.25, GH77) belongs to the type II 4-α-glucanotransferase family, and enzymes of this family have four known activities: disproportionation, cyclization, coupling, and hydrolysis.10−12 Amylomaltase was first found in E. coli and is one of the most important enzymes in the maltose system.6,13 Bacterial strains lacking amylomaltases are unable to utilize maltose and maltotriose as the sole source of carbon. 14,15 This enzyme was first described as a © XXXX American Chemical Society

Received: May 22, 2016 Revised: July 1, 2016 Accepted: July 1, 2016

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DOI: 10.1021/acs.jafc.6b02296 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. A pathway of maltose/maltodextrin and glycogen degradation in E. coli.3 The compounds with bold black font are the key metabolites of the pathway. Proteins and enzymes encoded by the genes shown are GlgP, glycogen phosphorylase; GlgX, glycogen debranching enzyme; MusI, a maltose transporter, MusEFGKI; Glk, glucokinase; α-Pgm, α-phosphoglucomutase; MalZ, maltodextrin glucosidase; MalP, maltodextrin phosphorylase; MalQ, 4-α-glucanotransferase. scaled using the HKL2000 software suite.34 The CgAM crystals belonged to the space group P212121, with unit cell parameters of a = 73.21 Å, b = 83.41 Å, and c = 125.32 Å. With one molecule of CgAM per asymmetric unit, the Matthews coefficient was approximately 2.45 Å3·Da−1, which corresponds to a solvent content of approximately 49.78%.35 The structure of CgAM was determined by molecular replacement with the CCP4 version of MOLREP36 using the structure of amylomaltase from E. coli (EcMalQ, PDB code 4S3P, 30% sequence identity) as a search model. The initial model building was performed automatically using ARP/wARP,37 the final model building was performed using the WinCoot program,38 and the refinement was performed with REFMAC5.39 The geometric parameters of the final model were validated using PROCHECK40 and MolProbity.41 The data statistics are summarized in Table 1. The refined model of CgAM was deposited in the Protein Data Bank with PDB code 5B68. Size-Exclusion Chromatographic Analysis. To investigate the oligomerization of CgAM, analytical size-exclusion chromatography was performed using a Superdex 200 10/300 column (GE Healthcare, 24 mL) at NaCl concentrations of 0, 50, 100, and 150 mM. 500 μL of protein samples with concentration of 2 mg/mL were analyzed. The molecular weights of the eluted samples were calculated based on the calibration curve of standard samples. To determine the void volume, we used blue dextran.

structure of CgAM at a resolution of 1.7 Å. We report the overall structure of CgAM and a structural comparison focusing on the N-domain of amylomaltases from different organisms. Furthermore, we will discuss the desultory oligomerization of CgAM and the key residues in its active site.

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MATERIALS AND METHODS

Cloning, Expression, and Purification. The CgAM gene was amplified by polymerase chain reaction (PCR) using genomic DNA from C. glutamicum strain ATCC 13032 as a template. The PCR product was then subcloned into pET30a, and the resulting expression vector pET30a:Cgam was transformed into the E. coli strain BL21(DE3)-T1R, which was grown in 1 L of lysogeny broth medium containing kanamycin at 37 °C. After induction by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside, the culture was further incubated for 20 h at 18 °C. The cells were then harvested by centrifugation at 4000g for 20 min at 4 °C. The cell pellet was resuspended in buffer A (40 mM Tris-HCl, pH 8.0 and 500 mM NaCl) and then disrupted by ultrasonication. The cell debris was removed by centrifugation at 13500g for 25 min, and the supernatant was applied to a Ni-NTA agarose column (Qiagen). After washing with buffer A containing 30 mM imidazole, the bound proteins were eluted with 300 mM imidazole in buffer A. Finally, trace amounts of contaminants were removed by size-exclusion chromatography using a Superdex 200 prep-grade column (320 mL, GE Healthcare) equilibrated with buffer A. All purification steps were performed at 4 °C. The degree of protein purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified protein was concentrated to 60 mg/mL in 40 mM Tris-HCl (pH 8.0) and 500 mM NaCl. The expression and purification of N-terminal-truncated CgAM were performed under the same conditions as those used for the native protein. Crystallization, Data Collection, and Structure Determination. Crystallization of the purified protein was initially performed with the following crystal screening kits: Index, PEG/Ion, and Crystal screen (Hampton Research) and Wizard I and II (Rigaku) using the hanging-drop vapor-diffusion technique at 20 °C. Each experiment consisted of 1.0 μL of protein solution and 1.0 μL of reservoir solution and then was equilibrated against 50 μL of the reservoir solution. The best quality CgAM crystals appeared in 2.0 M ammonium sulfate and 0.1 M bis-tris methane (pH 6.5). The crystals were transferred to a cryoprotectant solution containing 2.0 M ammonium sulfate, 0.1 M bis-tris methane (pH 6.5), and 30% (v/v) glycerol, extracted with a loop larger than the crystals, and flash-frozen by immersion in liquid nitrogen. Data were collected at 100 K at Beamline 7A at the Pohang Accelerator Laboratory (Pohang, Korea) with a Quantum 270 CCD detector (ADSC, USA). The data were then indexed, integrated, and

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RESULTS AND DISCUSSION Overall Structure of CgAM. To elucidate the molecular mechanism of amylomaltase from C. glutamicum ATCC 13032 (CgAM), we determined its crystal structure at 1.70 Å resolution (Table 1). The first three residues of the protein were invisible in the electron density map, and we modeled 703 amino acids (Arg4-Asp706) out of 706 amino acids. CgAM has overall folding similar to that of amylomaltase from E. coli (EcMalQ),33 and these two enzymes have amino acid identity of 30%. The CgAM structure consists of two distinctive domains: the N-terminal domain (N-domain, Met1-Arg165) and the C-terminal domain (C-domain, Leu166-Asp706) (Figure 2). The N-domain can be divided into two subdomains: subdomain I (N1-subdomain, Met1-Pro72) and subdomain II (N2-subdomain, Leu73-Arg165) (Figure 2B,C). The N1-subdomain consists of three α-helices and one doublestranded antiparallel β-sheet. The three α-helices form a helix bundle-like shape, and the β-sheet is attached to the C-domain side of the helix bundle. The N2-subdomain is composed of two four-stranded β sheets that form a β-sandwich. The N1subdomain interacts directly with the C-domain, whereas the B


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in the formation of the substrate-binding pocket, although the CA2-subdomain is the main contributor. Structural Comparison of CgAM with Other Amylomaltases. A Dali search42 showed that CgAM is structurally homologous to amylomaltases from E. coli (EcMalQ, PDB code 4S3P, Z-score 41.3), T. aquaticus (TaAM, PDB code 1CWY, Zscore 34.0), and T. thermophilus HB8 (TtAM, PDB code 2OWX, 33.1), and disproportionating enzymes (DPEs) from Arabidopsis thaliana (AtDPE1, PDB code 5CPQ, Z-score 32.0). Both amylomaltases and plastidal plant DPEs belong to the 4α-glucanotransferase type. To compare CgAM with homologous structures, we superposed the CgAM structure with those of EcMalQ, TtAM, and AtDPE1. Striking differences were observed at the N-domains of these proteins, whereas the overall structures of the C-domains matched each other closely (Figure 3A,B). The conspicuous disharmony between the Ndomains of CgAM and EcMalQ is related to the N2-subdomain and a long helix. The varying length of the β-strands in the nonconserved N2-subdomain results in different structures in this subdomain (Figure 3A,B). The N2-subdomain of CgAM showed much lower B-factors than that of EcMalQ. In addition, the linker between the N-terminal subdomains in EcMalQ is a flexible nine-residue loop, whereas that in CgAM is mainly occupied by the α-helix comprising 17 residues (Figure 3A,B). Most importantly, the N-domain is completely absent in thermostable TtAM. However, AtDPE1 has a long N-terminal arm region that stretches toward the neighboring subunit to form a dimer (Figure 3B).43 The biological and structural function of the N-domains in CgAM and EcMalQ are not yet clear. However, it is notable that the term β-sandwich has been used to explain the immunoglobulin fold, and the Greek key or jelly roll topology is often found in carbohydrate-binding modules (CBMs), as seen in various lectins.44−46 Dali server analysis also reveals various filamins and immunoglobulins with Z-scores of 7−8 as homologues of the N2-subdomain. To investigate the function of the N-domain of CgAM, we tried to express the N-domain-truncated form of CgAM. However, the truncated protein was found in the insoluble fraction, although the expression system was the same as that used by the wildtype protein (data not shown). We speculate that the decreased solubility of the N-domain-truncated form of CgAM is caused by exposure of the N-domain-binding interface of the Cdomain to the solvent. In fact, the N-domain-binding interface of the C-domain is mainly composed of nonpolar residues such as Val470, Ile483, Ile493, Leu496, and Leu500. Interestingly, TtAM that lacks an N-domain has a more hydrophilic surface at the corresponding region than CgAM (Figure 3C). Although the overall structures of the C-domains matched well, local structural differences were observed at two distinctive regions (Figure 3D). First, compared with other proteins, CgAM has a unique structure at the connecting loop between α8 and α9 in the CA1-subdomain, which is derived from the deletion of 20 amino acids at α8 (Figures 2A, 3D). Second, CgAM and EcMalQ contain an extended α17 and an extra α16 in the CA3-subdomain, which cause the formation of a bulged structure compared with TtAM and AtDPE1 (Figures 2A, 3D). These structural differences do not seem to directly influence the function of the enzymes because they occur at locations that are distal from the catalytic site. However, the regions are located at the edge of the substrate-binding site, especially when bulky or long polysaccharides are used as substrates, which might subsequently affect substrate specificity or the substrate binding mode of the enzymes (Figure 3D).

Table 1. Data Collection and Refinement Statistics CgAM apo PDB code

5B68 Data Collection

wavelength (Å) unit cell (a, b, c) (Å) space group solvent content (%) protein chains in AU resolution range (Å) highest resolution shell (Å) unique reflections redundancy completeness (%) Rmerge (%) av I/σ(I) Refinement resolution (Å) no. of reflections Rwork/Rfree no. of atoms protein bis-tris/glycerol/sulfate ion water mean B value (Å2) protein bis-tris/glycerol/sulfate ion water B from Wilson plot (Å2) rms deviations bond lengths (Å) bond angles (deg) Ramachandran Plotb most favored regions (%) additional allowed regions (%)

0.97934 73.0, 83.3, 124.8 P212121 49.8 1 50.00−1.70 1.73−1.70 81053 5.0 (2.9)a 96.2 (90.7) 6.8 (29.8) 34.7 (3.1) 50.00−1.70 77044 17.0/21.6 6193 5530 14/12/45 592 30.5 30.4 49.1/45.2/61.9 36.9 22.3 0.018 1.863 99.0 1.0

a Values in parentheses are for highest-resolution shell. bThe calculated Ramachandran plot generated using MolProbity.41

N2-subdomain is loosely attached to the C-domain by forming an independent fold (Figure 2B,C). The C-domain consists of four subdomains: one core subdomain, and three auxiliary subdomains (Figure 2B,C). The core subdomain (CCsubdomain) is located at the center of the C-domain and forms a (β/α)8-barrel (TIM barrel) fold where eight helices (α5, α9, α12−14, η9, α18, and α20) cover the eight-stranded βbarrel (β12−14, β17, and β20−23). The CC-subdomain forms a catalytic site and also contributes to the formation of the substrate-binding site, which will be described later. Three auxiliary subdomains form a triangle that covers the top of the CC-subdomain (Figure 2C). Auxiliary subdomain I (CA1subdomain; Met221−Trp358) comprises three 310-helices and three α-helices, and is located at the edge of (β/α)81 and (β/ α)82. Auxiliary subdomain II (CA2-subdomain; Val391−Gly439 and His461−Phe485) comprises two 310-helices and a threestranded twisted β-sheet, and is attached to (β/α)83 and (β/ α)84 of the CC-subdomain. The CA2-subdomain contacts the N-domain by interacting with the N1-subdomain. Auxiliary subdomain III (CA3-subdomain; Thr558−Gly625 and Leu650−Asn693) comprises two 310-helices and four α-helices, covers the edge of (β/α)87 and (β/α)88, and also contacts the CA1-subdomain. The auxiliary subdomains are heavily involved C


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Figure 2. Overall structure of CgAM. (A) Amino acid sequence alignment of amylomaltases from C. glutamicum (CgAM), E. coli (EcMalQ), Aquifex aeolicus (AaAM), T. thermophilus (TtAM), and disproportionating enzyme 1 from A. thaliana (AtDPE1). The secondary structure elements are marked on top of the alignment based on the CgAM structure: α-helices and 310-helices by a helix and β-strands by an arrow. The η symbol refers to a 310-helix. Conserved residues are boxed in white on a red background; similar residues are boxed in red with a white background. Residues involved in enzyme catalysis and maltose binding are indicated with triangles colored with red and blue, respectively. This figure was produced using ClustalW.48 (B) Domain identification of CgAM. N1 and N2 are representatives of N1- and N2-subdomains of N-domain, CC is core subdomain of C-domain, and CA1, CA2, and CA3 are auxiliary subdomains I, II, and III of C-domain, respectively. (C) Overall structure of CgAM. The structure of CgAM is presented as a cartoon diagram. The right side figure is rotated 90 deg in horizontal direction. A bis-tris molecule and a sulfate ion bound in CgAM are shown as sphere models and labeled. The subdomains of CgAM are distinguished with color scheme as in panel B. All structure images were produced using PyMOL.49

Oligomeric States. Oligomerization varies among amylomaltases and DPEs. It has been reported that although EcMalQ forms oligomers (dimers or tetramers) in 150 mM NaCl, the protein exhibits a monomeric state according to size-exclusion chromatography at an ionic strength of 500 mM NaCl.33 In the case of amylomaltase from Aquifex aeolicus (AaAM, PDB code 1TZ7), two different oligomeric conformations are predicted: one as a monomer by authors, and the other calculated by PISA (Protein Interactions, Surfaces and Assemblies) software (Figure 4A). As mentioned, AtDPE1 adopts a dimeric state involving the N-terminal arms, and its dimer corresponds to

two molecules in the AaAM tetramer. To investigate the oligomeric status of CgAM, we superposed our CgAM monomeric structure with the tetramer of AaAM and the dimer of AtDPE1 (Figure 4A). Interestingly, the N2-subdomain of CgAM hinders the formation of a tetramer like AaAM (Figure 4A). Moreover, the extended α17, a unique structural feature of CgAM and EcMalQ, hinders the adoption by CgAM of a dimeric form like AtDPE1 (Figure 4A). Because CgAM and EcMalQ contain similar N-terminal domains, we suspect that CgAM follows an oligomerization mode similar to that of EcMalQ. In fact, the crystallization of CgAM required 250 mM D


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Figure 3. Structural comparison of CgAM with EcMalQ, TtAM, and AtDPE1. (A) Amino acid sequence alignment of the N-domain of CgAM and EcMalQ. The secondary structure elements of the N-domain of CgAM and EcMalQ are marked on top and bottom of the alignment, respectively. (B) Comparison of N-domains of the proteins. Structures of CgAM, EcMalQ, TtAM, and AtDPE1 are superposed. C-domains of four proteins are shown with a gray color. N-domains of CgAM and EcMalQ are shown with colors of green and orange, respectively, and N-terminal arm region of AtDPE1 is shown with a cyan color. (C) Charge distribution on the surfaces of C-domains of CgAM and TtAM. The N-domain and the C-domain of CgAM are shown as a ribbon and an electrostatic potential surface mode, respectively (left), and TtAM is shown as an electrostatic potential surface mode (right). N-domain interacting region on the C-domain is indicated with a magenta-colored dotted line (left), and the corresponding region of TtAM is indicated as in CgAM. (D) Structural differences on C-domain. Structures of CgAM, EcMalQ, TtAM, and AtDPE1 are superposed. Cdomains of four proteins are shown with a gray color. N-domains of CgAM and EcMalQ are shown as in panel A. An AGA molecule bound in EcMalQ is shown as a stick model with a salmon color. The left side and the right side figures are magnified figures of two structurally different regions shown in the middle figure. Structurally different regions of CgAM, EcMalQ, TtAM, and AtDPE1 are shown in green, orange, magenta, and cyan colors, respectively. Key secondary structure elements located in the regions are labeled.

from structural and biochemical studies on EcMalQ33 and TtAM,26 where the invariant catalytic triad residues, two aspartates and one glutamate, are involved in enzyme catalysis. In EcMalQ, Asp448 acts as a base (the catalytic nucleophile) and Glu496 acts as a proton donor (acid/base catalyst) with the help of the third residue, Asp548 (transition state stabilizer). In the CgAM structure, the catalytic residues Asp460, Glu508, and Asp561 are located at the corresponding positions of Asp448, Glu496, and Asp548 in EcMalQ, which suggests that CgAM catalyzes the reaction by a mechanism similar to that in EcMalQ (Figure 5A). To elucidate the substrate-binding mode, we attempted to determine the structure of CgAM in a complex with various substrates such as maltose and maltoheptaose. However, neither cocrystallization with substrate nor soaking of the substrates into the CgAM crystal was successful. Nevertheless, superimposition of the CgAM structure with other amylomaltase structures bound with substrates enabled us to identify the substrate-binding mode of CgAM. We superposed CgAM with

NaCl and we did not observe oligomers from the noncrystallographic symmetry. The results imply that CgAM exists as a monomer in 250 mM NaCl. We further investigated the oligomeric status of CgAM using size-exclusion chromatography at various concentrations of NaCl (Figure 4B). CgAM was eluted as a monomer in 50, 100, and 150 mM NaCl (Figure 4B). However, the protein showed a dimeric peak without NaCl (Figure 4B). Based on these results, we conclude that the oligomeric status of CgAM is sensitive to NaCl concentration, and considering the physiological NaCl concentration of approximately 150 mM in bacterial cytosol,47 the protein might function as a monomer in bacterial cells. Taken together, unlike AtDPE1, most bacterial amylomaltases function as a monomer. Active Site of CgAM. At the active site of our current CgAM structure, one bis-tris methane molecule and one sulfate ion were bound (Figure 5A). These ligands mimic substrate recognition by CgAM, which will be described in detail later. The catalytic mechanism of amylomaltase has been proposed E


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EcMalQ complexed with maltose (PDB code 4S3Q) and acarviosine-glucose-acarbose (AGA) (PDB code 4S3R). In the accommodation of AGA in EcMalQ, seven subsites (−4 to +3) were identified with a catalytic site at the center (0) along each hexose moiety (Figure 5B). The donor site is located at the left side (subsites −4 to −1), whereas the acceptor site (subsites 1 to 3) position is at the right side when they are viewed from above the barrel with the lowest N-domain. In the maltosebinding mode of subsite −1 of EcMalQ, stabilizers Asp548 and His547 are heavily involved via hydrogen bonds with each of the hydroxyl groups of C2 and C3 of the glucose moiety, corresponding to Asp561 and His560 in CgAM, respectively (Figure 5C). In the EcMalQ−maltose complex, the other glucose moiety of maltose at subsite −2 is stabilized by hydrogen bonds with the main chain of Gly651 and Trp413 and the side chain of Asp548 and Asn648. CgAM has the same residues at corresponding positions, such as Trp425, Asp561, Asn661, and Gly664 (Figure 5C). As mentioned above, our current CgAM structure contains one bis-tris methane molecule and one sulfate ion bound at the active site (Figure 5A). The hydroxyl groups of bis-tris methane form hydrogen bonds with residues at the substrate-binding site, as do the hydroxyl groups of C2 and C3 from the glucose moiety in subsite −1 (Figure 5C). The sulfate ion exactly mimics the hydroxyl groups of C2 and C3 from the glucose moiety at subsite −2 (Figure 5C). Notably, the residues in subsites −2 to +2 in CgAM are completely identical to those in EcMalQ. This indicates that the recognition of maltose and short donors by CgAM might be similar to that by EcMalQ. However, Ala380 and Leu406 in EcMalQ located at the edge of the substrate-binding site are replaced by His392 and Tyr418 in CgAM. This implies that CgAM and EcMalQ might recognize a substrate in a mode that differs somewhat from that when long polysaccharides are used as a substrate. In addition, it is reported that the flexible lid (the 250s loop in TtAM and the 400s loop in EcMalQ) undergoes a conformational change upon substrate binding.20,33 In the CgAM structure, the flexible lid corresponds to the connecting loop between α15 and α16, and its amino acids are also highly conserved, indicating that CgAM also contains the flexible lid, which undergoes a conformational change upon substrate binding, as observed in TtAM and EcMalQ. Finally, we performed mapping of the conserved surface residues in several amylomaltases, and the highly conserved residues were mainly located at the active site, indicating that all the amylomaltases, including CgAM, catalyze the reaction in a similar way (Figure 5D). In summary, the CgAM structure revealed that CgAM exhibits a unique structural feature at the N-domain and also shows striking structural differences at the C-domain compared to other amylomaltase structures. Although functional implications of the N-domain still remain as terra incognita, the differences provide a structural basis for further application of the enzyme. It was reported that, when enzymatic reaction of CgAM was performed using pea starch as a substrate, degree of polymerization (DP) profiles of cyclodextrin products are from 19 to 50, with dominant DP from 27 to 28.27 Our observation on the differences at the extended edge of the substrate-binding site might explain why CgAM produces cyclodextrins with unique DP profiles compared to amylomaltases from other organisms. Thus, further studies on DP profiles of cyclodextrin depending on the edge region are required, and this observation might be useful for the modification of the industrially utilized enzymes.

Figure 4. Oligomeric status of CgAM. (A) Comparison in oligomerization of CgAM, AtDPE1, and Aquifex aeolicus AM (AaAM). A CgAM monomer, a AtDPE1 dimer, and a AaAM tetamer are superposed, and shown as cartoon diagrams. The N-domain and the C-domain of CgAM are shown with green and magenta colors, respectively. The N-terminal arm and the C-domain of AtDPE1 are shown with cyan and orange colors, respectively. AaAM is shown as a gray color. (B) Size-exclusion chromatographic analysis of CgAM. The elution peaks from NaCl concentration of 0, 100, and 150 mM are indicated with arrows, and calculated molecular weights and oligmeric status are labeled. For precise analysis of the molecular weight, standard samples of ferritin (440 kDa), aldolase (158 kDa), ovalbumin (44 kDa), and ribonuclease A (13.7 kDa) are used for calibration and labeled 1−4, respectively. F


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Figure 5. Active site of CgAM. (A) Electron density map of a Bis-tris molecule and a sulfate ion bound at the active site pocket. A simulated annealing composite omit map50 of a bis-tris molecule and a sulfate ion bound at the active site pocket are shown with blue-colored mesh and contoured in 3 σ. The structures of CgAM and EcMalQ are superposed, and three catalytic residues are shown with green- and cyan-colored sticks, respectively. The polar contacts between the ligands and the residues are shown with red-colored dotted lines, and the distances are labeled. (B) Substrate binding pocket of CgAM. The CgAM structure and the EcMalQ structure in complex with AGA are superposed. Subsites based on AGA are labeled (−4 to +3). The CgAM structure is shown as a surface model, and the AGA molecule bound in EcMalQ is shown as an orange-colored line. The bis-tris methane molecule and the sulfate ion bound in CgAM are shown as stick models and labeled. One catalytic residue, D561, is shown as a green-colored stick. (C) Stabilization of the bis-tris methane molecule and the sulfate ion. The CgAM structure and the EcMalQ structure in complex with maltose are superposed. The bis-tris methane molecule is shown as a stick with a magenta color, and the sulfate ion is shown as a stick with a yellow color. The maltose molecule bound in EcMalQ is shown as a salmon-colored stick. Residues involved in the stabilization of the bis-tris methane molecule and the sulfate ion in CgAM are shown as green-colored sticks, and their corresponding residues in EcMalQ are shown as cyancolored sticks. Bis-tris methane molecule and the sulfate ion bound at the catalytic site are shown as sticks with magenta and yellow colors, respectively. (D) Surface conservation of amylomaltases. The CgAM structure is shown as a surface conservation model. Highly conserved residues are shown as a stick model and labeled. Mapping of the conserved residues on the surface of CgAM was performed using ClustalW48 and ConSurf51 programs. For amino acid comparison, six amylomaltase family enzymes including C. glutamicum (CgAM), E. coli (EcMalQ), A. aeolicus (AaAM), T. aquaticus (TaAM), T. thermophilus (TtAM), and A. thaliana (AtDPE1) are used.

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*Tel: +82-53-950-5377. Fax: +82-53-955-5522. E-mail: kkim@ knu.ac.kr. Author Contributions ‡

S.J. and S.K. contributed equally to this work.

Funding

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIP) (2014R1A2A2A01005752 and 2014M1A2A2033626), and was also supported by the New & Renewable Energy Core Technology Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) granted financial resource from the Ministry of Trade, Industry & Energy, Republic of Korea (No. 20153030091360). Notes

The authors declare no competing financial interest. G


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Crystal Structure of Amylomaltase from Corynebacterium glutamicum

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