Structure and mechanism of MalQ

Amylomaltase MalQ is essential for the metabolism of maltose and maltodextrins in Escherichia coli. It catalyzes transglycosylation/disproportionation reactions in which glycosyl or dextrinyl units are transferred among linear maltodextrins of various lengths. To elucidate the molecular basis of transglycosylation by MalQ, we have determined three crystal structures of this enzyme, i.e. the apo form, its complex with maltose and an inhibitor complex with the transition state analog acarviosine-glucoseacarbose, at resolutions down to 2.1 Å. MalQ represents the first example of a mesophilic bacterial amylomaltase and exhibits an Nterminal extension of about 140 residues, in contrast with previously described thermophilic enzymes. This moiety seems unique to amylomaltases from Enterobacteriaceae and folds into two distinct subdomains that associate with different parts of the catalytic core. Intriguingly, the three MalQ crystal structures appear to correspond to distinct states of this enzyme, revealing considerable conformational changes during the catalytic cycle. In particular, the inhibitor complex highlights the requirement of both a 3-OH group and a 4-OH group (or α1–4glycosidic bond) at the acceptor subsite +1 for the catalytically competent orientation of the acid/base catalyst Glu496. Using an HPLCbased MalQ enzyme assay, we could demonstrate that the equilibrium concentration of maltodextrin products depends on the length of the initial substrate: with increasing number of glycosidic bonds less glucose is formed. Thus, both structural and enzymatic data are consistent with the extremely low hydrolysis rates observed for amylomaltases and underline the importance of MalQ for the metabolism of maltodextrins in E. coli. Uptake and metabolism of α1–4-linked glucose polymers in E. coli is accomplished by the maltose system (1). Within the cell, maltose/maltodextrins are mutually converted by the three enzymes MalP, MalQ and MalZ, while MalP and MalQ are the major players for the catabolic degradation of maltodextrins. MalP cleaves glucose moieties by phosphorolysis from the non-reducing end to yield glucose-1-P, with maltopentaose constituting the shortest substrate (4,5). Cells lacking MalP accumulate large amounts of linear maltodextrins, due to the action of MalQ (6). MalQ converts short maltodextrins, including maltose and maltotriose, to longer maltodextrins and glucose. Bacterial strains lacking MalQ cannot grow on maltose and maltotriose (7). Concerted action of MalP and MalQ via maltodextrins as common intermediates leads to the formation of glucose http://www.jbc.org/cgi/doi/10.1074/jbc.M115.667337 The latest version is at JBC Papers in Press. Published on July 2, 2015 as Manuscript M115.667337 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Structure and mechanism of MalQ 2 and glucose-1-P. Both are subsequently converted to glucose-6-P by glucokinase and phosphoglucomutase, respectively, to enter glycolysis. Increased internal glucose levels appeared to reduce the activity of MalQ (8). Furthermore, maltodextrin metabolism is linked to that of glycogen via the shared intermediate maltotriose, which is not only a substrate of MalQ but also constitutes the inducer of the maltose system (9,10). Thus, degradation of glycogen leads to a basal induction of mal genes even in the absence of external maltodextrins. On the other hand, in the presence of maltose and maltotriose, MalQ can compensate a lack of glycogen synthase GlgA (11). MalQ cleaves any linear maltodextrins, releases their reducing end and transfers the resulting non-reducing dextrinyl moiety (donor) onto another maltodextrin or glucose (acceptor) (12,13). This results in a series of maltodextrins that differ in length by one monosaccharide unit as well as glucose (Fig. 1). MalQ from E. coli was the first amylomaltase to be isolated and biochemically characterized (12-15). Much later, the malQ gene was cloned and its encoded amino acid sequence of 694 residues (78.5 kDa) was elucidated (16). MalQ is a member of the amylomaltases (EC 2.4.1.25) or 4-αglucanotransferases, which are structurally and mechanistically related to α-amylases. Both belong to the glycoside hydrolase clan H (GHH). However, based on sequence characteristics, amylomaltases have been classified as a separate GH family (GH77) (17). In contrast to α-amylases, which mostly catalyze hydrolysis of glycosidic bonds, amylomaltases strongly favor the transglycosylation or disproportionation reaction with retention of the α-anomeric configuration (18). Structures of amylomaltases from three Thermus species were previously described (1921). Notably, with ~500 residues the thermophilic homologues are considerably shorter than MalQ from E. coli. The larger part of the additional ~190 residues forms a ~140residue N-terminal extension of hitherto unknown structure and function. Here we present three distinct crystal structures of MalQ from E. coli: the apo form, the complex with maltose and the complex with the heptasaccharide transition state analog acarviosine-glucose-acarbose (AGA), which represent different states of the catalytic cycle. Furthermore, we have employed an HPLC-based assay to monitor the MalQ product spectrum at equilibrium, revealing the dependence on the number of glycosidic bonds in the substrate(s). EXPERIMENTAL PROCEDURES Enzyme cloning, expression and purification The C-terminal ten residues of MalQ (UniProt ID P15977) represent an unusual repeat of amino acids, i.e. Arg4Ala3Lys3, which were most likely structurally disordered. To decrease the flexibility and positive charge at the C-terminus of MalQ, the six C-terminal residues were replaced by SerAlaHis6, thus appending a His6tag for affinity purification. To this end, the malQ gene was amplified from the genomic DNA of the E. coli strain XL1-blue by PCR with Pfu Ultra II DNA polymerase (Agilent) using the primer pair 5'-GA GAT ATA CAT ATG GAA AGC AAA CGT CTG GA-3' / 5'-GCT TCT GCG CCG TCT GTC CAA ATC CTT CAG CAA-3' and cloned on pASK75-his (22) via NdeI and AfeI (Fermentas). The resulting expression vector encodes the 78.9 kDa protein MalQ(1-688)-SAHHHHHH (MalQ-His) under the control of a tetracycline-dependent promoter. The correct sequence of the vector was confirmed by DNA sequencing. MalQ-His was produced in the E. coli strain BL21 in LB medium. To incorporate Lselenomethionine (SeMet) into the recombinant protein for experimental phasing of the diffraction data, bacteria were cultivated in M63 medium (22 mM KH2PO4, 40 mM K2HPO4, 15 mM (NH4)2SO4, 0.2 % (w/v) glucose, 1 mM MgSO4, 4.5 μM FeSO4) supplemented with 50 mg/L L-SeMet (Acros Organics), 0.02 mg/L tryptophan, 0.4 mg/L of the other proteinogenic amino acids as well as 0.04 mg/L 4aminobenzoic acid, 0.04 mg/L 4-hydroxybenzoic acid, 0.4 mg/L xanthine, 0.4 mg/L uracil, 0.01 mg/L biotin, 0,01 mg/L nicotinamide, 1 μg/L riboflavin and 0.01 mg/L thiamine. Bacteria were cultivated at the 2 L scale using shake flasks at 22 °C. Protein expression was induced by adding 0.2 mg/L anhydrotetracycline at OD550 = 0.6. After 6 h (16 h for SeMet incorporation), the cells were harvested by centrifugation and the pellets were stored at -20 °C. After thawing on ice, the bacteria were resuspended in 0.5 M NaCl, 50 mM HEPES/Na pH 8.0, 10 mM imidazole and 2 mM βmercaptoethanol (BME) and disrupted in a French pressure cell (Thermo Fisher Scientific). The cell suspension was cleared by centrifugation at 35000 g and the supernatant was applied onto an IDA-Sepharose column by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Structure and mechanism of MalQ 3 charged with Zn ions. Unbound protein was rinsed with 0.5 M NaCl, 50 mM HEPES/Na pH 8.0, 2 mM BME, then MalQ-His was eluted with a linear gradient of 0–0.3 M imidazole in the same buffer. Appropriate fractions were pooled, dialyzed against 0.5 M NaCl, 20 mM Tris/HCl pH 8.0, 2 mM BME and further purified by size exclusion chromatography (SEC) using a preparative Superdex200 26/60 column (GE Healthcare) equilibrated with the same buffer. SEC fractions corresponding to monomeric MalQ were pooled, supplemented with 2 mM tris(2-carboxethyl)phosphine (TCEP) and concentrated to 13 mg/ml for crystallization. The final yield per liter culture was 12 mg MalQ. Protein crystallization, data collection and refinement – Crystals of MalQ-His were obtained by sitting drop vapor diffusion at 20 °C using an Evo robotic system (Tecan). Drops containing 200 nl protein and 200 nl reservoir solution were equilibrated against 100 μl reservoir. Crystals of apo-MalQ formed in the presence of 1.2 M Na3citrate. Complexes of MalQ with maltose (MalQ•maltose) and acarviosine-glucose-acarbose (MalQ•AGA) were obtained by incubation of MalQ-His with 2 mM maltotriose (Applichem) and 2 mM acarbose (LKT Laboratories, Inc.), respectively, prior to crystallization. Diffraction quality crystals of MalQ•maltose grew in the presence of 20 % (w/v) PEG-3350, 0.2 M Kl, whereas MalQ•AGA and SeMet-derivatized MalQ•AGA crystals were obtained in 20 % (w/v) PEG-3350, 0.2 M NaH2PO4 and 20 % (w/v) PEG-3350, 0.2 M NaCl, respectively. Cryoprotection of the crystals for flash freezing in liquid nitrogen was achieved by adding 3 μl of the corresponding reservoir solution supplemented with 25 % (v/v) ethylene glycol, 0.25 M NaCl and 10 mM Tris/HCl pH 8.0. X-ray diffraction data were collected at beamline 14.2 of the BESSY synchrotron (Berlin-Adlershof, Germany) operated by the Helmholtz-Zentrum Berlin (23). Data sets were processed with the XDS package (24) (Table 1). Crystallographic phases were determined by multiple wavelength anomalous dispersion (MAD) with a SeMet-derivatized MalQ•AGA crystal. To this end, datasets at four different wavelengths, e.g. high-energy remote, peak, inflection point and low-energy remote, were collected (Table 1). Phases were determined with HKL2MAP (25). All 21 expected selenium sites could be identified, resulting in a well interpretable electron density map. The initial structure was automatically built with BUCCANEER (26) and completed in repeating cycles of manual model building with COOT (27) and refinement with REFMAC5 (28) (Table 1). Translation, libration and screw (TLS) groups were determined with TLSMD (29) and used during refinement with REFMAC5. Subsequently, the structures of apo-MalQ and MalQ•maltose were solved by molecular replacement with the refined MalQ•AGA structure using PHASER (30). Again, model building and refinement were accomplished with COOT and REFMAC5. Structures were validated with COOT and MolProbity (31). Molecular graphics were prepared with PyMOL (Schrödinger). Atomic coordinates and structure factors have been deposited in the Protein Data Bank (www.rcsb.org/pdb) with accession codes 4S3P, 4S3Q, and 4S3R. Enzyme assay – A MalQ enzymatic assay was performed with the substrates maltose (Applichem), maltotriose as well as the inhibitor acarbose. Also, different ratios of maltose/glucose and maltotriose/glucose were investigated. Each sugar was dissolved at 0.1 M in 100 μl 0.5 M NaCl, 20 mM Tris/HCl pH 8.0, 2 mM BME. The reaction was started at 30 °C by adding 1.5 μl of a 1.3 mg/ml solution of MalQ in the same buffer. All setups were performed in triplicate. Reactions were stopped at different time points between 3 min and 16 h by inactivation of MalQ at 95 °C for 3 min. Denatured enzyme was removed by centrifugation and the supernatant was mixed with 100 μl trichloroacetic acid and 200 μl 1% (w/v) dansyl hydrazine in ethanol and incubated at 80 °C for 10 min (32). 5 μl of the resulting sugar derivatives were injected into a 1200 HPLC system (Agilent, Santa Clara, CA) equipped with a VP 260/10 NUCLEODUR C18 column (Machery-Nagel, Dueren, Germany). Elution of the sugar hydrazones was performed with a gradient of 10–30 % (v/v) acetonitrile in 10 mM NH4HCO3 pH 8.5 and monitored at 340 nm. The C18 column was calibrated with dansyl hydrazones of glucose, maltose, maltotriose and acarbose. Obviously, dansyl hydrazones of maltotetraose and maltopentaose could also be resolved on this column. The retention times of the sugar hydrazones were 23.6, 22.4, 20.7, 20.6, 18.6 and 17.2 min for glucose, maltose, maltotriose, acarbose, maltohexaose and maltopentaose, respectively. The corresponding peak areas were integrated and the relative by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Structure and mechanism of MalQ 4 amount of each sugar was plotted against time with Kaleidagraph software (Synergy Software). Statistical simulation of the maltodextrin product profile from the MalQ reaction – Maltodextrin equilibrium concentrations were calculated by means of a contingency table comprising all possible MalQ reactions up to maltodecaose using Microsoft Office Excel 2007 (see Supplementary Information, Table S1). For practical reasons, the set of donor and acceptor molecules was limited to a maximal length of ten glucose units. Attack of each glycosidic bond within a donor oligosaccharide was considered equally likely, only for glucose as donor no reaction was assumed. In each cell of the table the maltodextrin product concentration was calculated as the mathematical product of donor and acceptor concentrations divided by the number of glycosidic bonds of the donor (to account for the equal distribution of possible reaction products). The resulting maltodextrin product concentrations were summed up for each chain length. These sums were then used as starting concentrations for the next round of calculation. Final equilibrium concentrations were obtained after ten iterations (cf. Table S1). RESULTS Purification and structure determination of MalQ – After overexpression in E. coli MalQ was purified to homogeneity by IMAC and SEC. Attempts to transfer MalQ into a buffer of moderate ionic strength, i.e. 150 mM NaCl or less, resulted in soluble oligomers of the enzyme as judged by SEC. Therefore, MalQ was always kept in a buffer containing 500 mM NaCl during purification, crystallization and enzymatic assays (cf. Materials and Methods). Crystals of apoMalQ, MalQ•maltose, MalQ•AGA belonged to space groups P2221, P1, and P212121, respectively, containing 2, 3 and 1 polypeptide chains per asymmetric unit (Table 1). Both chains of apo-MalQ showed a continuously interpretable electron density for residues 2–690 including the residues Ser and Ala of the linker between the protein and Cterminal His6-tag. In addition, molecule 1 showed density for the N-terminal Met residue as well as the first His of the tag. The MalQ•maltose complex was obtained by cocrystallization of MalQ with maltotriose. The asymmetric unit contained three polypeptide chains, which showed electron density for residues 2–43 and 52–690, whereas residues 44– 51 were disordered in all three chains. One and five C-terminal His residues of the affinity tag were observed in molecules 1 and 2, respectively. In the following, we will refer to protein chain 1 of each asymmetric unit for apoMalQ and the MalQ•maltose complex, respectively. MalQ crystallized in the presence of acarbose showed interpretable electron density for residues 2–42 and 52–690. Again, the connecting residues 43–51 were not resolved whereas unambiguous density was visible for a ligand (see below). Overall structure of MalQ – The tertiary structure of MalQ can be subdivided into three major domains: A, B and N (Fig. 2). Domain A represents the catalytic core and adopts a TIM barrel fold (33), comprising a central barrel of 8 parallel β-strands, each flanked by an α-helix, in a counter-clockwise manner. Noteworthy, in MalQ the sixth α-helix is missing and replaced by a 310-helix with a single turn. The (β/α)8 core harbors several insertions at the carboxyl-end of the β-strands. Together, these insertions are referred to as domain B (21) and comprise the three distinct subdomains B1–B3 (Fig. 2). Contrasting with previous reports, these subdomains are here sequentially numbered. B1 represents the largest of the three subdomains, with 166 residues. It shows a purely α-helical architecture and is a characteristic feature of the amylomaltase family GH77 (17,21). B1 comprises residues 186–351, which are inserted after strand β2 of domain A (Aβ2). Basically, this subdomain can be viewed as a pair of 4-helical bundles that wrap around the Nterminal part of helix Aα2. The functionally most important subdomain is B2, which forms a lid for the active site during catalysis and harbors the 400s loop (250s loop in the thermophilic homologues; see further below). In MalQ, this subdomain consists of two insertions after strands Aβ3 and Aβ4 and includes residues 377427 (B2a) and 449-473 (B2b). B2 shows a mixed α/β structure with a central four-stranded, antiparallel twisted β-sheet flanked by short αhelices. B2 interacts with B1 via the two helical segments of B2a, which precede the first βstrand. Finally, two shorter helical insertions, forming subdomain B3 and comprising residues 545–613 (B3a) and 637–672 (B3b) are found downstream of strands Aβ7 and Aβ8, respectively. B3a carries the transition statestabilizing side chain of Asp548. Subdomain B3 interacts with B1 via B3a but does not form any by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Structure and mechanism of MalQ 5 contacts with B2 in the absence of a substrate. In contrast, substrate binding induces conformational changes that lead to extensive contacts between B3a and B2a, as seen in the MalQ•AGA structure. A short insertion occurs after strand Aβ1, where it binds at the interface of B1 and B3. The longer loop that follows Aβ6, which interacts with B3a, is a result of the missing helix Aα6 mentioned above. In addition to the catalytic domains A and B, MalQ exhibits a peculiar N-terminal extension of 128 residues, referred to as domain N. These residues show no significant sequence homology to other known protein structures. This extension is present in most Enterobacteraceae but missing in other γProteobacteria. Domain N folds into the subdomains N1 (1–42) and N2 (52–128), both connected by a flexible nine-residue linker that is only resolved in the apo-MalQ structure (Fig. 2). Subdomain N1 comprises two helices – connected by a short two-stranded antiparallel βsheet – that pack against each other at an angle of 52°. This subdomain tightly associates with the two insertions of subdomain B2 and thus undergoes the same domain movements as B2 in the course of the catalytic cycle (see below). Notably, the αββα topology of N1 is similar to that of B2a (residues 390–425) (cf. Fig. 2). Nevertheless, due to the structural flexibility and the low sequence identity of this motif (12 % over 40 residues), it is difficult to infer an evolutionary relationship. Subdomain N2 resembles a β-sandwich consisting of a five-stranded mixed β-sheet and a short two-stranded antiparallel β-sheet. N2 is rather loosely attached to domain A via its Cterminal portion (c.f. Fig. 2). At first glance, the N2 structure is reminiscent of carbohydratebinding modules (CBMs). However, a comparison with structurally known CBMs did not reveal significant similarities. In fact, CBMs that bind α1–4-linked sugars (tribes CBM-A to C) (34) show different topologies. N-terminal extensions similar to N2 of MalQ were also observed in other sugar metabolizing enzymes, such as cellulase CelA from Alicyclobacillus acidocaldarius (PDB code 3H2W) (35) or the structurally and mechanistically related maltosyl transferase GlgE from Streptomyces coelicolor (PDB code 4C4N) (36), but could not be attributed to a discrete function. Basically, subdomain N2 adopts a fibronectin type III (Fn)-like fold (37). A distinct feature of subdomain N2 is a variation of the characteristic Fn β-sandwich of two antiparallel β-sheets abe and c'cfg, in which βstrand a associates with β-strand g of the latter sheet in a parallel manner. Comparison of the crystal structures of MalQ in three different ligand states – In the crystal structure of apo-MalQ the active site did not show any electron density for a ligand molecule. Residues of the catalytic triad comprising the nucleophile Asp448, the acid/base catalyst Glu496 and the putative transition state stabilizer Asp548 interact with other residues of the active site pocket: Asp448 forms a salt bridge with Arg446, Glu496 is hydrogen-bonded to the main chain nitrogens of His449 and Leu498, and Asp548 is hydrogenbonded to Asn648. In contrast, interpretable ligand density was observed in the substrate pocket of molecule A of the MalQ•maltose complex, which was in accordance with a maltose molecule that occupies subsites -2 and -1 (38) (Fig. 3). Apparently, the observed maltose resulted from the MalQ-catalyzed conversion of maltotriose, the compound that was actually added to the crystallization solution (see Materials and Methods and further below). Crystallographic B values and occupancy refinement of the maltose suggest a ligand occupancy of about 70 %. Overall, the substrate pocket is slightly narrower than in the apo structure (Fig. 4). Binding of the first glucose moiety to subsite -2 involves two hydrogen bonds with Asn648 and a water molecule, as well as van der Waals contacts with Pro650 and Gly651. The second glucose in subsite -1 is hydrogen-bonded to Asp548 and His547 and also undergoes hydrophobic interactions with Trp413 and Tyr201 (Fig. 3A). Notably, with a distance of 4.2 Å the catalytic nucleophile Asp448 is too far away for a potential attack of C1 in subsite -1. Also, the acid/base catalyst Glu496 and the putative transition state stabilizer Asp548 are with 6.0 Å and 4.8 Å too distant from the O1 atom in subsite -1. Thus, the MalQ•maltose complex represents a catalytically incompetent conformation. Surprisingly, in the crystal structure of MalQ obtained with the tetrasaccharide ligand acarbose electron density revealed seven connected carbohydrate moieties in the active site. This observation can be explained by a MalQ-catalyzed conversion of two acarbose molecules to the elongated – and even more by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Structure and mechanism of MalQ 6 potent – inhibitor acarviosine–glucose–acarbose (AGA) upon release of one glucose molecule (see further below). The excellent density and the B values of AGA after refinement indicate 100 % occupancy of this heptasaccharide analog. However, the B values of the individual sugar units are not uniform. At subsites -2 to +2, the sugar moieties show the lowest B values, with an average of ~33 Å, followed by subsites -3 and +3 with ~41 Å. The highest B value with 65 Å was observed for the cyclohexene moiety at subsite -4, i.e. at the periphery of the substrate