Cell. Mol. Life Sci. (2016) 73:2643–2660 DOI 10.1007/s00018-016-2243-9 Cellular and Molecular Life Sciences

MULTI-AUTHOR REVIEW

Distribution of glucan-branching enzymes among prokaryotes

1 1 Eiji Suzuki • Ryuichiro Suzuki

Received: 21 April 2016 / Accepted: 22 April 2016 / Published online: 3 May 2016 Ó Springer International Publishing 2016

Abstract Glucan-branching enzyme plays an essential role Abbreviations in the formation of branched polysaccharides, glycogen, AGPase ADP-glucose pyrophosphorylase and amylopectin. Only one type of branching enzyme, BE Branching enzyme belonging to glycoside hydrolase family 13 (GH13), is CAZy Carbohydrate-active enZymes found in eukaryotes, while two types of branching enzymes CBM Carbohydrate-binding module (GH13 and GH57) occur in prokaryotes (Bacteria and CSR Conserved sequence region Archaea). Both of these types are the members of protein DBE Debranching enzyme families containing the diverse specificities of amylolytic DP Degree of polymerization glycoside hydrolases. Although similarities are found in the GH Glycoside hydrolase catalytic mechanism between the two types of branching GS Glycogen synthase enzyme, they are highly distinct from each other in terms GT Glycosyltransferase of amino acid sequence and tertiary structure. Branching LGT Lateral gene transfer enzymes are found in 29 out of 30 bacterial phyla and 1 out MGLP Methylglucose lipopolysaccharide of 5 archaeal phyla, often along with glycogen synthase, SBS Surface/secondary binding site suggesting the existence of a-glucan production and stor- SS Starch synthase age in a wide range of prokaryotes. Enormous variability is observed as to which type and how many copies of branching enzyme are present depending on the phylum Introduction and, in some cases, even among species of the same genus. Such a variation may have occurred through lateral trans- Glucan-branching enzyme (abbreviated as BE; EC fer, duplication, and/or differential loss of genes coding for 2.4.1.18) catalyzes the formation of a-1,6-bonds in the branching enzyme during the evolution of prokaryotes. branched polysaccharides, glycogen in animals and microorganisms, or amylopectin (the major component of Keywords Alpha-glucan Á Branching enzyme Á Bacteria Á starch) in plants [1, 2]. The frequency and position of the Archaea Á Glycoside hydrolase Á Glycosyltransferase Á branch points are important determinants for the structure Carbohydrate-active enzymes and properties of these reserve carbohydrates, and BE plays a pivotal role in specifying these characteristics [3, 4]. The BE reaction proceeds in two steps: first, a preexisting a- 1,4-glucan chain is cleaved and the non-reducing portion of the donor chain is covalently attached to carboxyl group of & Eiji Suzuki [email protected] the catalytic residue (Asp in GH13 BE or Glu in GH57 BE; see below) at the active site of the enzyme [5, 6]. The 1 Department of Biological Production, Akita Prefectural glucan moiety is then transferred to the C-6 hydroxyl group University, 241-438, Kaidobata-Nishi, Shimoshinjyo- of the same or another glucan chain (the acceptor chain) Nakano, Akita 010-0195, Japan 123 2644 E. Suzuki, R. Suzuki

[4]. Through this double displacement mechanism, the a- classification system of carbohydrate-active enzymes based configuration of anomeric carbon is retained, resulting in on amino acid sequence similarities (http://www.cazy.org/) the formation of a-1,6-linkage. If a water molecule instead was founded, the a-amylase family proteins were classified of the C-6 hydroxyl group of a sugar is involved in the into GH family 13 [15]. As the number of members latter step of the reaction, the enzyme serves as a hydrolase increased considerably, the GH13 family was subdivided rather than a transferase. Therefore, BE is mechanistically into 35 subfamilies [16] (currently expanded to 40 sub- similar to amylolytic enzymes. BEs are actually homolo- families). Until now, BEs have been classified into only gous to a-amylases and grouped into glycoside hydrolase two subfamilies, GH13_8 and GH13_9, that include BEs in (GH) families (although only a limited number of BEs Eukaryotes and Bacteria, respectively (although with show hydrolytic activity). exceptions as described below). Notable distinction has been recognized concerning the Apart from the identification and expansion of GH13 a- distribution of BE in eukaryotes and prokaryotes: BEs in amylase family, a-amylases with distinct sequences were animals, fungi, and plants show higher sequence similar to described in a thermophilic bacterium, Dictyoglomus each other than to BEs in bacteria [7, 8]. This situation is in thermophilum [17] and a thermophilic archaeon Pyrococ- sharp contrast to some of the other enzymes responsible for cus furiosus [18]. Both of these enzymes, currently starch metabolism in plants, including ADP-glucose considered to be 4-a-glucanotransferase (EC 2.4.1.25) [19, pyrophosphorylase (AGPase), starch synthase (SS), and 20], were classified into the GH57 family [21]. Together debranching enzyme (DBE). The latter enzymes in plants with a-amylase and 4-a-glucanotransferase, GH57 family were acquired from bacteria during endosymbiosis that also contains enzymes with other catalytic specificities, gave rise to plastids, and do not have homologs in animals amylopullulanase (EC 3.2.1.41), and even a-galactosidase or fungi (in the case of AGPase and DBE), or they show (EC 3.2.1.22) [22]. Finally, a GH57 protein with unknown much higher similarities to those in bacterial rather than function in a thermophilic archaeon, Thermococcus animal/fungal counterparts (SS) [8]. In contrast to rather kodakaraensis, was identified as BE [23]. Homologs of T. uniform distribution of one type of BE in eukaryotic lina- kodakaraensis GH57-type BE were found to occur in ges, two distinct types of BE, one being moderately related several linages of Bacteria (Firmicutes, Actinobacteria, and to the eukaryotic counterpart, and the other showing little Cyanobacteria) [23]. Two types of BE (belonging to GH13 sequence similarity to eukaryotic BEs, have been found and GH57) may have derived from distinct origins and across two Domains of prokaryotes, Bacteria and Archaea. have undergone convergent evolution to show the common According to ‘‘List of Prokaryotic Names with Standing catalytic function along with the related enzymes involved in Nomenclature’’ (http://www.bacterio.net), the Domains in modification, and degradation of a-glucans. Bacteria and Archaea are divided into 30 and 5 phyla (or divisions), respectively, and most of these phyla have representative organisms with completed genomes. In this Primary and tertiary structures of glucan- review, current knowledge of structural and functional branching enzymes features of BEs, and distribution of BEs in bacterial and archaeal phyla are described. Domain composition and sequence motifs of BEs are schematically represented in Fig. 1. Both GH13 and GH57 BEs are multidomain proteins. GH13 BEs consist of, from Occurrence of two distinct types of glucan- N to C termini, carbohydrate-binding module (CBM) 48, branching enzyme central catalytic domain referred to as domain A, and domain C. In some GH13_9 BEs, CBM48 is further pre- The primary structure of BE was first elucidated in ceded by domain N, which is generally absent from Escherichia coli [9]. It was noted that the deduced amino GH13_8 BEs. Indeed, GH13_9 BEs are divided into group acid sequence of BE showed similarity to those of amy- 1 and group 2, depending on the presence or absence, lolytic enzymes [10–12]. The amylolytic enzymes include respectively, of domain N [24] (Fig. 3). Domain N, a-amylase (EC 3.2.1.1), pullulanase (EC 3.2.1.41), CBM48, and domain C consist of approximately 100 isoamylase (EC 3.2.1.68), and cyclomaltodextrin glucan- amino acid residues, and all these domains adopt a b- otransferase (EC 2.4.1.19), in addition to BE, thus enzymes sandwich fold [25, 26]. CBM48 is also found in related catalyzing all four types of reaction; transfer and hydrolysis protein of GH13 (referred to as pullulanase subfamily) of a-1,4- and a-1,6-linkages [13]. They shared some con- including pullulanase (GH13_12, GH13_13, and served sequence regions (CSRs), thus collectively GH13_14), isoamylase (GH13_11), and maltooligosyltre- constituting a protein family, which was designated as a- halose trehalohydrolase (EC 3.2.1.141, GH13_10) [27]. amylase family [13, 14]. When CAZy database, a The central domain A is composed of a (b/a)8 barrel 123 Distribution of glucan-branching enzymes among prokaryotes 2645

100 amino acids (a) GH13_8 Clostridium DE (AEV69892) VI IVII III IV VII

(b) GH13_9 1 1 2 23 3 4 4 566 7 78 8 Escherichia DE (AAA23872) VI IVII III IV VII Bacillus DE (AAC00214) VI IVII III IV VII β α domain N CBM48 ( / )8 barrel (domain A) domain C

(c) GH57 1 1 2 53 6 4 779 101011 Thermococcus ED

(BAD85625) 1 23 D 45 HhH HhH Thermus ED (BAD71725) 1 23 D 45 Bacillus ED E (BAB05134) 1 2 3D 4 5 GT4

domain B N-do- β α ( / )7 barrel (domain A) domain C N-domain C-domain main

Fig. 1 Domain organization and sequence motifs of branching composed of (b/a)7 barrel domain (domain A), an a-helical domain enzymes in prokaryotes. a A GH13_8 BE from Clostridium (domain B, striped) inserted between b2 and a5 in domain A, and clariflavum DSM 19732 (AEV69892). b GH13_9 BEs from C-terminal a-helical domain (domain C, stippled). Conserved Escherichia coli (AAA23872) and Bacillus subtilis (AAC00214). sequence regions 1–5 [32] and an additional conserved region D The GH13 BEs consist of domain N, CBM48 (cyan), central (b/a)8 [23] are indicated by dark hatched bars and light hatched bars, barrel domain (domain A), and domain C (red)[25, 26, 38, 56]. respectively. Positions of catalytic residues glutamate (E) and aspartic Consensus sequence regions I–IV [28] and additional conserved acid (D) are shown in red. Loop regions found in Thermococcus and sequence regions V–VII [31]in(b/a)8 barrel domain are indicated by Thermus BEs are indicated by wavy lines. Additional sequences are dark hatched bars and light hatched bars, respectively. Positions of attached to some GH57 BEs: (1) HhH, helix–hairpin–helix, (2) GT4, catalytic residues aspartate (D) and glutamic acid (E) are shown in glycosyltransferase family 4 consisting of N- and C-domains [37], red. Secondary structures in (b/a)8 barrel domain are indicated by with putative catalytic residue glutamate (E) shown in green triangles (b strands, numbered in green) and rectangles (a helices, (inconsistency in the domain nomenclature exists, e.g. domain N numbered in gray). c GH57 BEs from Thermococcus kodakarensis vs. N-domain, as they originated from different studies). Secondary KOD1 (BAD85625), Thermus thermophilus HB8 (BAD71725), and structures in (b/a)7 barrel domain are indicated as in (b). All proteins Bacillus halodurans C-125 (BAB05134). The GH57 BEs are are drawn to scale although a5 is not obvious between b5 and b6 (Fig. 1) A and domain C [32]. When GH57 BE of T. kodakaraensis [25]. Four conserved sequence regions (CSRs) I to IV are was initially characterized, 6 CSRs (A through F) were located at b strands (b3, b4, b5, and b7, respectively) and proposed [23]. Among them, CSRs A, B, C, E, and F their succeeding loops [28] (Figs. 1, 2). The two essential corresponded to CSRs 1, 2, 3, 4, and 5, respectively [32], residues at the active site, the nucleophile aspartate and the while CSR D was found specifically in BE, but not in the acid/base glutamic acid are located in CSRs II and III, other enzymes of GH57 family. The catalytic residues respectively [5, 14, 29, 30]. Among other conserved resi- glutamate (nucleophile) and aspartic acid (acid/base) are dues, another aspartate is located in CSR IV, and serves to located in CSRs 3 and 4, respectively [6]. Extra loop stabilize the reaction intermediate [5]. Additional CSRs V, regions were found between b7 and b9 in a limited number VI, and VII have been proposed [31]. The CSRs VI and VII of BEs including those from T. kodakaraensis [33] and correspond to b strands (b2 and b8, respectively) and the Thermus thermophilus [6] (Fig. 4). Some members of following loops, whereas the CSR V is located at a loop GH57 BEs have extra sequence at their C termini: (1) facing the end of CSR I, constituting a wall of the active Repeated helix–hairpin–helix (HhH) motif was reported in site cleft [26]. GH57 BE from T. kodakaraensis [23]. The (HhH)2 GH57 BEs are composed of a (b/a)7 barrel domain (also sequence also occurs in other GH57 BEs (if not all) from referred to as domain A), an a-helical domain (domain B) Thermococcales archaea. (2) Some GH57 BEs from Fir- inserted between b2 and a5 of domain A, and another a- micutes (Bacilli and Clostridia) are fused to helical domain (domain C) following domain A [6] glycosyltransferase (GT) family 4 sequence at the C ter- (Fig. 1). Five CSRs (1 through 5) are distributed in domain minus. GT4 is the second largest GT family in the CAZy

123 2646 E. Suzuki, R. Suzuki

(a) (c)

(b) (d)

E183 D405 E458 D354

(e) 150 (f) 0 Eco 120 3 Eco

) Mtu

90 Tth 6 Tth Tko 60 9 Depth (Å) Tko

Width (degree 30 12 Mtu

0 15 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Rotation (degree) Rotation (degree)

Fig. 2 Overall architecture and geometry around the active site of were depicted using PyMOL [89]. e Width of the active site. The BEs. a GH13_9 BE from Escherichia coli (PDB ID = 1M7X) [25]. width was defined as the angle between two lines connecting the CBM48 in cyan, (b/a)8 domain in gray, central b-strands in green, catalytic residue and atoms located on the opposite rims of the active domain C in red. b Magnification around the active site of BE from site cleft. The plane of the section was then rotated by 30° and the E. coli. Aspartate (D405) serving as the catalytic nucleophile and calculated width was plotted against the angle of rotation. The angles glutamic acid (E458) serving as the acid/base are shown in ball and were represented as a function of the rotation angle. Eco Escherichia stick representation. c GH57 BE from Thermococcus kodakaraensis coli (1M7X), Mtu Mycobacterium tuberculosis (3K1D) [26], Tko (PDB ID = 3N8T) [33]. The (b/a)7 domain in gray, domain B Thermococcus kodakaraensis (3N8T), Tth Thermus thermophilus inserted between b2 and a5of(b/a)7 domain in cyan, central b- (3P0B) [6], f depth of the active site, calculated as the distance strands in green, a loop region above the active site in yellow between the line connecting the two atoms on the opposite rims of the (corresponding to the yellow wavy line in Fig. 1c), domain C in red. d active site cleft, and the catalytic residue. The plane of the section was Magnification around the active site of BE from T. kodakaraensis. rotated by 30° and the calculated depth was plotted against the angle The catalytic nucleophile glutamate (E183) and the acid/base aspartic of rotation acid (D354) are in ball and stick representation. Crystal structures database (next to GT2) and contains 48,000 members, lobes of Rossmann fold [36] indicated as N- and C-do- represented by nearly 20 different enzyme specificities. In mains in Fig. 1c (the last segment of the protein is folded terms of tertiary structure, proteins in GT4 family adopt the as a part of the N-domain) [37]. A glutamate, putative GT-B fold, one of the two major folds in glycosyltrans- catalytic residue of GT-B fold enzymes is conserved, but ferase families, along with other GT families, GT3, GT5, enzymatic specificity conferred by the GT4 domain of this and GT35 [34, 35]. The GT-B fold proteins consist of two fusion protein remains to be elucidated. As glycogen

123 Distribution of glucan-branching enzymes among prokaryotes 2647 synthases (GSs) belonging to GT4 family have been relationship between tertiary structure of the enzyme and identified in some bacteria (Table 1), it is tempting to substrate specificity, therefore, remains unsolved. speculate that the GH57-GT4 protein has BE and GS The catalytically critical residues are located on the activities responsible for glycogen synthesis. However, the concave surface of the enzyme. The geometry around the organisms containing this fusion protein also have GS catalytic residues is compared between GH13 and GH57 independently (Table 1). BEs by calculating the ‘‘width’’ and ‘‘depth’’ of the active Overall architecture and geometry around the active site site. As shown in Fig. 2e and f, the active site cleft is wider of BEs are shown in Fig. 2. Crystal structure of GH13_9 and shallower in GH13 BEs than in GH57 BEs. The BE has been elucidated for E. coli [25, 38] and My- structural distinction around the active site may signifi- cobacterium tuberculosis [26]. Based on these structures, cantly affect the specificity of the enzyme. However, GH13-type BE exhibits an elongated structure, as CBM48, comparative biochemical characterization of BE across domain A, and domain C are arranged linearly along a different families has not been extensively performed [6]. longitudinal axis of the protein. The structure and the arrangement of domain N in the E. coli BE have not been solved, while in M. tuberculosis BE, domain N was located Catalytic specificity of branching enzymes at the far end of the enzyme preceding CBM48, and the whole protein adopts a highly elongated shape [26]. The Determination of the catalytic property of BE is essential to catalytic residues aspartate and glutamic acid (indicated as understand the mechanism, by which the molecular struc- D405 and E458, respectively, in Fig. 2b) are located in ture of the reserve polysaccharide is formed. However, the adjacent loops (immediately following b4 and b5 strands) enzymatic characterization of BE is not very simple, as the [25, 26]. Binding of oligosaccharides was observed at action of BE is tightly coupled with the elongation of the several sites in the crystal structure of E. coli BE [38]. glucan chain by glycogen/starch synthase. In the in vitro These binding sites can be regarded as surface/secondary assays, artificial substrates (e.g. commercial amylose and binding sites (SBSs) [39], in which oligosaccharides are amylopectin) are generally used, which may or may not bound at a certain distance of the active site. SBSs have well reproduce the actual situation in vivo. Nevertheless, a been described for a wide range of glycoside hydrolases number of studies have been carried out to characterize the [39], and are particularly prominent in GH13 [40]. Crystal catalytic specificity of BEs, mostly belonging to GH13 structures have also been reported for BEs from rice (one family (Fig. 3). Comparative characterization of bacterial of three isoforms, BEI) [41, 42] and human [43]. The and eukaryotic BEs has been reported [44, 45]. structure of these eukaryotic BEs, belonging to GH13_8 In many instances, BE specifically shows a-1,6-trans- subfamily, is similar to those of bacterial counterparts, ferase activity (as opposed to a-1,4-transferase or a-1,4- except for the presence of a-helical segment at the N ter- hydrolase activities). Hydrolase activity is thus below the minus instead of domain N, and the arrangement of two detection limit for BEs from Arthrobacter globiformis [46], loops (b3-a3, and b4-a4) in domain A [41, 43]. Butyrivibrio fibrisolvens [47], Deinococcus geothermalis Crystal structures of GH57 BE were elucidated for the and D. radiodurans [48], Anaerobranca gottschalkii [49], enzymes from Thermococcus kodakaraensis [33] and Vibrio vulnificus [50]. In contrast, BE from Streptococcus Thermus thermophilus [6]. In these structures, two a-heli- mutans showed a-1,4-hydrolytic activity in addition to a- cal domains (domains B and C) are folded together with the 1,6-branching activity [51]. A GH57 BE in Thermus ther-

(b/a)7 barrel domain (domain A), so that the whole protein mophilus showed hydrolase activity, which was 4 % of the exhibits a compact, triangular shape. The central b strands total activity (measured as the amount of reducing ends constitute an incomplete barrel structure by two distorted before and after isoamylase treatment of the reaction pro- sheets, arranged as b10–b1–b2–b3 and b4–b7–b9, with a duct, respectively) [6]. Besides a-1,6-transferase activity, gap between b9 and b10 [6, 33]. The catalytic residues thermostable BEs from Rhodothermus marinus [52] and R. glutamate and aspartic acid (indicated as E183 and D354, obamensis [53] showed activity of a-1,4-transferase [52, respectively, in Fig. 2d) are from loops following b4 and 53] and a-1,4-hydrolase [52]. b10 strands, respectively, and are located at the opposite Concerning the substrate specificity, BEs from A. sides of the active site pocket [6, 33] (Fig. 2). In GH57 BE, globiformis [46], B. fibrisolvens [47], and S. mutans [51] subsites were predicted based on a binding of modeled showed activities both on amylose and amylopectin. The maltotriose [6], or on the positions of glucose and glycerol BE activity of D. geothermalis was much higher on amy- located around the active site [33]. lose than amylopectin, while the activity of D. radiodurans In spite of a number of studies as described above, the was higher on amylopectin than amylose [48]. R. oba- binding of oligosaccharides at the active site cleft has not mensis BE was six times more active on amylose than been observed in any of the BE crystal structures. The amylopectin, whereas it showed little activity on glycogen 123 2648 E. Suzuki, R. Suzuki

Table 1 Distribution of BE and GS in prokaryotes BE Total number of GS GH13_8 GH13_9 GH57 GH13 GH57 GT3 GT5 GT4a

Bacteria Acidobacteria Ca. Solibacter usitatus Ellin6076 - d -81-d Ca. Chloracidobacterium thermophilum - - d 21-d Actinobacteria Corynebacterium glutamicum ATCC 13032 - d -70--d Frankia alni ACN14a - d -100 --d Streptomyces coelicolor A3(2) - dd -180 --d Frankia sp. EuI1c - dd91--d Mycobacterium tuberculosis H37Rv - dd71--d Conexibacter woesei DSM 14684 - - d 01--- Saccharomonospora viridis DSM 43017 - - d 71--- Aquificae Aquifex aeolicus VF5 - d -11-d Thermovibrio ammonificans HB-1 - - d 03-d Armatimonadetes (OP10) Chthonomonas calidirosea T49 - - d 22-- Fimbriimonas ginsengisoli Gsoil 348 - - d 51-d Bacteroidetes Bacteroides fragilis 638R d --61ddb Cytophaga hutchinsonii ATCC 33406 d --41ddb Tannerella forsythia ATCC 43037 dd --51ddb Flavobacterium johnsoniae UW101 - d -90-ddb Sphingobacterium sp. 21 - dd -100 -ddb Caldiserica (OP5) Caldisericum exile AZM16c01 - - d 03-d Chlamydiae Chlamydia trachomatis D/UW-3/CX - d -20-d Chlorobi Chlorobaculum tepidum TLS - - - 6 0 - d Chlorobium limicola DSM 245 - - - 6 0 - d Ignavibacterium album JCM 16511 ? ? - 8 0 - dd Chloroflexi Thermobaculum terrenum ATCC BAA-798 - d -70-- Chloroflexus aurantiacus J-10-fl - ddc 16 2 - - d Herpetosiphon aurantiacus ATCC 23779 - - dd 12 2 - dd Chrysiogenetes (NKB19) Desulfurispirillum indicum S5 - - d 03-d Cyanobacteria Prochlorococcus marinus CCMP1986 - dd31-d Synechococcus elongatus PCC 7942 - dd21-d Cyanothece sp. PCC 8802 - dd d 12 2 - dd Cyanothece sp. ATCC 51142 - ddd d 15 3 - dd Deferribacteres Denitrovibrio acetiphilus DSM 12809 - - d 03-dd Deinococcus-Thermus Deinococcus radiodurans R1 - d -110 -d

123 Distribution of glucan-branching enzymes among prokaryotes 2649

Table 1 continued BE Total number of GS GH13_8 GH13_9 GH57 GH13 GH57 GT3 GT5 GT4a

Thermus thermophilus HB8 - - d 42-d Dictyoglomi Dictyoglomus thermophilum H-6-12 ATCC 35947 - - d 14-- Elusimicrobia (Termite group I) Elusimicrobium minutum Pei191 - - d 12-d Fibrobacteresc Fibrobacter succinogenes S85 - dd33-d Firmicutes Clostridium clariflavum DSM 19732 d - -20-dd Lactococcus lactis CV56 d - -80-d Selenomonas ruminantium TAM6421 dd-50-d Bacillus subtilis subsp. subtilis str. 168 - d -80-d Clostridium botulinum B str. Eklund 17B - d -120 -d Lactobacillus casei BD-II - d -80-d Clostridium perfringens SM101 - dd -90-d Paenibacillus sp. JDR-2 - dd -50-dd Ruminococcus obeum A2-164 - ddd -90-d Clostridium kluyveri DSM 555 - dd42-d Paenibacillus mucilaginosus 3016 - dd10 1 - d Bacillus halodurans C-125 - - d 71-d Clostridium acetobutylicum ATCC 824 - - d 41-d Acetohalobium arabaticum DSM 5501 - - dd 12-d Desulfotomaculum kuznetsovii DSM 6115 - - dd 23-d Fusobacteria Fusobacterium nucleatum ATCC 25586 - d -20-d Leptotrichia buccalis C-1013-b - - d 51-d Gemmatimonadetes Gemmatimonas aurantiaca T-27 - d -11-dd Lentisphaerae Lentisphaera araneosa HTCC2155 - d -??--d Candidate phylum NC10 Ca. Methylomirabilis oxyfera - d -12-d Nitrospirae Ca. Nitrospira defluvii - d -21-d Leptospirillum ferrooxidans C2-3 - - d 53-d Planctomycetes Planctomyces brasiliensis DSM 5305 - d -41-d Phycisphaera mikurensis NBRC 102666 - - d 32d - Proteobacteria a-Proteobacteria Paracoccus denitrificans PD1222 - d -20-d Rhodobacter sphaeroides 2.4.1 - d -100 -d Rhizobium leguminosarum bv. trifolii WSM1325 - dd -110 -d Rhodobacter sphaeroides ATCC 17025 - dd -110 -d Magnetospira sp. QH-2 - dd43-ddd b-Proteobacteria Burkholderia multivorans ATCC BAA-247 - d -70--

123 2650 E. Suzuki, R. Suzuki

Table 1 continued BE Total number of GS GH13_8 GH13_9 GH57 GH13 GH57 GT3 GT5 GT4a

Thiobacillus denitrificans ATCC 25259 - d -41-d Sulfuricella denitrificans skB26 - dd -82-d Variovorax paradoxus B4 - dd -90-d c-Proteobacteria Acidithiobacillus ferrooxidans ATCC 23270 - d -32-d Escherichia coli MG1655 - d -100 -d Vibrio cholerae IEC224 - d -60-d Moritella viscosa - dd -70-d Xanthomonas oryzae KACC 10331 - dd -90-d Methylomicrobium alcaliphilum 20Z - dd d 11 3 - dd Nitrosococcus halophilus Nc 4 - dd d 10 2 - dd d-Proteobacteria Desulfovibrio desulfuricans ATCC 27774 d --11d - Desulfovibrio vulgaris Miyazaki F - d -21-d Desulfovibrio africanus Walvis Bay - dd -72-d Sorangium cellulosum So ce56 - dd11 1 - dd Myxococcus xanthus DK 1642 - dd11 1 - d e-Proteobacteria Nitratifractor salsuginis DSM 16511 - d -12-d Sulfurimonas autotrophica DSM 16294 - d -12-dd Spirochaetes Brachyspira pilosicoli WesB - - d 13-dd Spirochaeta thermophila DSM 6578 - - d 43-d Treponema pallidum Nichols - - dc02-- Synergistetes Thermanaerovibrio acidaminovorans DSM 6589 - d -11-d Tenericutes Mycoplasma mobile 163 K - d -40-d Thermodesulfobacteria Thermodesulfatator indicus DSM 15286 - - d 03-d Thermotogae Petrotoga mobilis SJ95 - dd92-d Thermotoga maritima MSB8 - - d 61-d Verrucomicrobia Akkermansia muciniphila ATCC BAA-835 d - -31-- Opitutus terrae PB90-1 - d -110 -d Methylacidiphilum infernorum V4 - dd74-dd Archaea Crenarchaeota Aeropyrum pernix K1 ---00-- Pyrobaculum aerophilum IM2 - - - 1 3 - d Sulfolobus solfataricus P2 ---32-d Euryarchaeota Archaeoglobus fulgidus DSM 4304 - - - 0 0 - - Halobacterium sp. NRC-1 - - - 0 0 - - Methanocaldococcus jannaschii DSM 2661 - - - 0 1 - d Methanoculleus bourgensis MS2 - d -31--

123 Distribution of glucan-branching enzymes among prokaryotes 2651

Table 1 continued BE Total number of GS GH13_8 GH13_9 GH57 GH13 GH57 GT3 GT5 GT4a

Picrophilus torridus DSM 9790 - d -31-- Thermococcus kodakaraensis KOD1 - - d 44-d Presence or absence of BE and GS belonging to different (sub)-families is shown. Bullet (•) and hyphen (-) indicate presence and absence of the particular type of the enzyme, respectively. The number of bullets represents the number of isoforms. Total number of GH13 and GH57 members in organisms are also shown. The phyla are arranged in alphabetical order a Only GSs in Actinobacteria and those showing high similarity to the actinobacterial proteins are indicated. Comprehensive analysis of the entire GT4 family has not been performed b Fragment sequences lacking the essential amino acid residues for catalysis c These proteins have a substitution at the catalytic amino acid residue

[53]. Among three isoforms of GH13_9 BE in Cyanothece BE3 produced short (DP 5–11) as well as long (DP 30–35) sp. ATCC 51142, two isoforms (BE1 and BE2) showed glucan chains [54, 56]. BEs of Aquifex [48, 57, 58] and higher activity on amylose than amylopectin, while the E. coli [44, 45, 48, 50] primarily transferred much longer activity of BE3 was much lower but comparable on amy- chains of DP 10–14. As the GH57 enzymes, BE in Ther- lose and amylopectin [54]. The activity of V. vulnificus BE mococcus kodakaraensis produced glucans of DP 5–30 was ten times higher with amylopectin than amylose [50]. with two local maxima at DP 6 and DP 11 [23], whereas E. coli BE showed higher activity on amylose than amy- BE in Thermus transferred glucan chains of DP 4–16 with a lopectin [55], which was opposed by a different study clear preference at DP 6 [6]. Based on the catalytic prop- reporting that E. coli BE had higher activity on amy- erty, BEs from various bacteria and eukaryotes were lopectin than amylose [50]. R. marinus BE showed a-1,6- classified into three types, corresponding to three BE iso- branching, a-1,4-disproportionating, and a-1,4-hydrolytic forms of rice: BEI producing short (DP 6–15) and long (DP activities on various substrates including amylose, starch, 26–39) glucan chains, BEIIa predominantly producing pullulan, and maltooligosaccharides as short as maltotriose short chains (DP 6 and 7) as well as longer chains (DP (the mode of catalysis differed depending on the substrate) 8–15), and BEIIb exclusively producing short chains (DP 6 [52]. A GH57 BE in T. thermophilus was active on amy- and 7) [44, 45, 54, 56, 59]. BE isoforms of rice hardly lose, showed only hydrolytic activity (and no transferase produce glucan chains shorter than DP 6 [59], and some activity) on amylopectin, and was virtually inactive on bacterial enzymes (e.g. V. vulnificus BE that transfers very glycogen [6]. short chains with DP B5 at high rates [50]) apparently The preferred lengths of glucan chains transferred by BE deviate from the classification scheme. are also variable depending on the source organism. V. Other aspects on the catalytic specificity of BEs have vulnificus BE transferred very short chains of degree of been demonstrated using elaborate experimental tech- polymerization (DP) 3–5, comprising greater than 20 % of niques, i.e., treatment of substrate or product all chains transferred [50]. R. obamensis BE also trans- polysaccharides by exoglycolytic digestion using phos- ferred short glucan chains of DP 3–8 [51]. D. geothermalis phorylase or b-amylase. (1) BEs from E. coli [44, 45], and D. radiodurans BEs transferred glucan chains of DP Synechococcus elongatus [44, 45, 56], and three isoforms 4–17 (predominantly DP 6–7) [48]. B. fibrisolvens BE gave of GH13_9 BE from Cyanobacterium sp. NBRC 102756 rise to branches of DP 5–10 (with a maximum peak at DP [56] showed activity on phosphorylase-limit dextrin, which 7) [47]. S. mutans BE primarily transferred oligosaccha- uniformly had short branches with four glucose residues rides of DP 6–7 [51]. R. marinus BE produced branched from the branch points. The result suggested that these BEs glucan from amylose, with the most abundant chains of DP transferred glucan chains bearing branch(es), provided that 12 [52]. Incubation of amylose with A. gottschalkii BE these enzymes could not attack short linear chains of DP resulted in the production of glucan chains estimated to be B4. This property was also observed for BEI isoform of ranging from DP 6 to 60 [49]. Two isoforms of BE (BE1 rice, but not for the other eukaryotic BEs examined [44, and BE2) in Cyanobacterium sp. NBRC 102756 and 45]. (2) Treatment of reaction products of BEs from E. coli Cyanothece sp. ATCC 51142 produced glucans of DP 6–7 [45]orS. elongatus [45, 60] by phosphorylase [45]orb- and DP 10 (as a major and minor peak, respectively), while amylase [60] for analysis of chain length profile indicated

123 2652 E. Suzuki, R. Suzuki

Flavobacterium ABQ05767 Runella AEI48415 Sphingobacterium ADZ81628** Bacteroidetes Sphingobacterium ADZ78735** Desulfovibrio EGJ51276** δ-Proteobacteria Sulfuricella BAN36234** Azoarcus CAL94413** β Azoarcus CAL94342** -Proteobacteria Variovorax AGU49213** Methylacidiphilum ACD83079 Verrucomicrobia Thermodesulfovibrio ACI20861 Nitrospirae Thermanaerovibrio ACZ18600 Synergistetes Waddlia CCB90682 Chlamydiae Aquifex AAC06895 [48,57,58] Aquificae Rhodothermus ACY48769 [52,53] Bacteroidetes Methanoculleus CCJ37194 Euryarchaeota_Archaea Chloroflexus ABY34391 Chloroflexi Candidatus Nitrospira CBK43150 Nitrospirae Nitrosococcus ADE15574** γ-Proteobacteria Candidatus Kuenenia CAJ73819 Planktomycetes Nitratifractor ADV46244 ε-Proteobacteria Slackia ACV22726 Arthrobacter CBT76115 [46] Actinobacteria Paracoccus ABL72492 Rhodobacter ABP70034** Ketogulonicigenium ADO43074 Mesorhizobium BAB54018 α-Proteobacteria Rhizobium ACS57893** Rhodobacter ABP70676** Acetobacter BAI00203 Azotobacter ACO78971 γ-Proteobacteria Rhizobium ACS59312** α-Proteobacteria Herbaspirillum ADJ63826** Herbaspirillum ADJ64002** β-Proteobacteria Burkholderia AJY15155 Xanthomonas AAW73448** Xanthomonas AAW73367** Escherichia AAA23872 [9,24,25,38,44,45,50,55] γ-Proteobacteria Yersinia AEL71888 Haemophilus AAC23004 Variovorax AGU47625** β-Proteobacteria Moritella CED60351** γ-Proteobacteria Magnetospira CCQ74842 α-Proteobacteria Rubrobacter ABG03292 Actinobacteria Moritella CED61099** γ Vibrio AFC59536 [50] -Proteobacteria Gemmatimonas BAH39752 Gemmatimonadetes Sorangium CAN94657 δ Myxococcus ABF92763 -Proteobacteria Streptomyces (I) CAA58314** [64,65] Streptomyces (II) CAB92878** [64,65] Frankia CAJ64527 Corynebacterium BAB98617 [63] Actinobacteria Mycobacterium CAA98090 [26,66,67] Amycolicicoccus AEF41678** Amycolicicoccus AEF39795** Cyanothece ACB51598*** [54] Synechococcus ABB57115 [44,45,56] Prochlorococcus CAE19043 Cyanobacteria Cyanothece ACB53943*** [54] Cyanothece ACB51156*** [54] Caldilinea BAL98309 Chloroflexi Isosphaera ADV63716 Planctomycetes Candidatus Methylomirabilis CBE69957 Phylum NC10 Solibacter ABJ88246 Acidobacteria Toxoplasma AAU01393 Alveolata_Eucarya Methylomonas AEF99311** Methylomonas AEF99935** γ-Proteobacteria Nitrosococcus ADE14269** Magnetococcus ABK43903** α Magnetococcus ABK44027** -Proteobacteria Nitrosomonas NE2029 β Sulfuricella BAN35737** -Proteobacteria Acidithiobacillus ACK80548 γ-Proteobacteria Thiobacillus AAZ98011 β-Proteobacteria Planctomyces ADY62044 Planctomycetes Desulfovibrio EGJ48505** δ Desulfocapsa AGF79707 -Proteobacteria Fibrobacter ACX76683 Fibrobacteres Opitutus ACB77118 Verrucomicrobia Chlamydia AAC68464 Chlamydiae Deinococcus AAF11402 [48] Deinococcus/Thermus Petrotoga ABX32021 Thermotogae Lentisphaera WP_007281050 Lentisphaerae Picrophilus AAT42652 Euryarchaeota_Archaea Thermobaculum ACZ41249 Chloroflexi Ruminococcus CBL22762*** Ruminococcus CBL23687*** Firmicutes Ruminococcus CBL24631*** Fusobacterium AAL95052 Fusobacteria Mycoplasma AAT27887 Tenericutes Clostridium ACD24131 Firmicutes Atopobium ACV51479 Actinobacteria Bacillus AAC00214 [50] Paenibacillus ACS99722** Paenibacillus ACT00914** Lactobacillus AEA57676 Firmicutes Streptococcus BAL68915 [51] Selenomonas BAL82148 Oryza BEI BAF20543 (GH13_8, outgroup) [12,41,44,45,59] Archaeplastida_Eucarya 0.2

Fig. 3 Maximum likelihood tree (constructed using PHYLIP [90]) indicate the number of GH13_9 isoform in these organisms. Solid for GH13_9 BEs. Proteins are indicated by the Genus name of the circles and open circles indicate nodes supported by bootstrap values source organism and GenBank accession number. Bar shows 0.2 of [75 and [50 %, respectively. Triangles indicate that these BEs substitutions per sequence position. Group 1 BEs that have domain N have been characterized are in green. Group 2 BEs devoid of domain N are in blue. Asterisks

123 Distribution of glucan-branching enzymes among prokaryotes 2653 that these enzymes formed the a-1,6-linkages preferentially Proteobacteria, and Verrucomicrobia). GH57 family, cur- at the second or third glucose residue from the reducing rently consisting of 1,335 members, is exclusive to end of the acceptor glucan chain. In contrast, BEs from prokaryotes, and includes 410 and 21 BEs in Bacteria and plants (including green or red algae), human, and yeast, all Archaea, respectively. It should be emphasized here that belonging to GH13_8, formed branch points with lower most of these BEs (without triangles in Figs. 3 and 4) specificities in terms of the distance from the reducing end remain to be characterized. Particularly, only three mem- [45, 60]. bers of GH57 BE and none of GH13_8 BE (from BEs are multidomain proteins, and roles of non-catalytic prokaryotes) have been experimentally investigated. domains in determining reaction specificity have also been Distribution of GH13_9 and GH57 BEs in prokaryotes is investigated. Truncation at the N terminus of E. coli BE by summarized in Table 1. BEs are found in many prokaryotic 112 amino acid residues (corresponding to the removal of phyla, but their distribution (as to which type and how domain N) resulted in the preference for transfer of longer many copies are present) is extremely variable within and glucan chains (from DP 8–14 to 15–20) [61]. Exchange of between phyla. Such variability is observed even between domains derived from BEs in D. geothermalis and D. species in the same genus (Table 1). A phylogenetic tree radiodurans showed that CBM48, but not domain C based on the amino acid sequences of GH13_9 BEs influenced substrate specificity (preference on amylose or (Fig. 3) shows that the primary structure of BEs does not amylopectin) and branching pattern (preferred lengths of necessarily reflect taxonomy. In some cases, BEs from a glucans transferred) [48]. Domain N of Vibrio vulnificus single organism or from organisms in the same taxa are BE was shown to determine the preference toward amylose located in distant branches of the tree, and in other cases, over amylopectin, and transfer of very short chains [50]. As BEs from organisms in distantly related taxa are found a comparison, experiments with chimeric proteins of maize close to each other (e.g. Actinobacteria and Proteobacte- BEI and BEIIb (both belonging to GH13_8) showed that ria). These observations suggest that the evolution of BE C-terminal portion of these BEs (a part of domain A after involves ancient gene duplication and diversification, lat- CSR IV and domain C) determined the substrate preference eral gene transfer (LGT), and/or differential gene loss. (amylose or amylopectin), whereas the central portion and/ or N terminus (CBM48 and a part of domain A before CSR Distribution of BE in individual phyla I) influenced the length of the chains transferred [62]. So far, no obvious correlation has been found between In Actinobacteria (Gram-positive bacteria with high G ? C the primary structure and catalytic specificity of BE [45]. It content), Corynebacterium glutamicum [63] and Frankia may be possible that the enzyme specificity of BEs is alni have one GH13_9 BE. Species in the genus Strepto- substantially altered by slight changes in the geometry myces have two BEs belonging to GH13_9. In around the active site (e.g. presence of a loop, or a specific Streptomyces coelicolor A3(2), two BEs (GlgBI and amino acid side chain contributing to form the substrate GlgBII) are differentially expressed in distinct cell types binding site) rather than the entire amino acid sequence of (in the boundary of substrate and aerial mycelia, and aerial the protein. hyphae, respectively), and the expression of the latter was controlled by WhiG (a sigma factor rwhiG responsible for the developmental regulation of sporulation) [64, 65]. The Distribution of glucan-branching enzymes above Actinobacteria do not have any member of GH57 in Bacteria and Archaea family. In contrast, Frankia sp. EuI1c and Mycobacterium tuberculosis [66, 67] have one each of GH13_9 and GH57 Overview BE. Conexibacter woesei and Saccharomonospora viridis have BE as the single member of GH57 family in these As of October 2015, the GH13 family contains 28,024 organisms. members, of which 2994, accounting for more than 10 % In Bacteroidetes, some species unusually contain of the whole GH13 family, are GH13_9 BEs in Bacteria. ‘‘eukaryotic type’’ GH13_8 BE. Bacteroides fragilis and Compared to the large number in Bacteria, only three each Cytophaga hutchinsonii have one GH13_8 BE, while of the GH13_9 BEs are found in Archaea (in the phylum Tannerella forsythia has two GH13_8 BEs. Flavobac- Euryarchaeota) and Eukaryotes (apicomplexan parasites). terium johnsoniae has one GH13_9 BE, whereas While 488 members of GH13_8 BE are in Eukaryotes, Sphingobacterium sp. 21 has two GH13_9 BEs. mostly represented by animals, fungi, and plants, 103 The phylum Chlorobi includes green sulfur bacteria members of GH13_8 BE (dubbed hereafter as ‘‘eukaryotic performing anoxygenic photosynthesis. Neither GH13 nor type BE’’) are also found in a limited number of bacterial GH57 BE has been found in Chlorobaculum tepidum TLS taxa (in the phyla Bacteroidetes, Firmicutes, d- and Chlorobium limicola DSM 245, although glycogen 123 2654 E. Suzuki, R. Suzuki

Caldisericum BAL81216 Caldiserica Clostridium AEB75573 Heliobacterium ABZ85498** Firmicutes Paenibacillus AFC27622 Sulfurihydrogenibium ACN98567 Aquificae Thermovibrio ADU96949 Denitrovibrio ADD67231 Deferribacteres Desulfurispirillum ADU66696 Chrysiogenetes Thermotoga AHD18669 [73,74] Thermotogae Leptospira ABZ94586 Spirochaetes Brachyspira CCG55670 Elusimicrobium ACC97615 Elusimicrobia Leptotrichia ACV38302 Fusobacteria Candidatus Chloracidobacterium ABV27192 Acidobacteria Cyanothece ACB51102 Synechococcus ABB57919 Cyanobacteria Prochlorococcus CAE19524 Synechococcus CAE07244 Methylacidiphilum ACD83633 Verrucomicrobia Myxococcus ABF87741 δ-proteobacteria Ammonifex ACX51582** Desulfotomaculum AEG15246** Moorella ABC20215** Firmicutes Thermincola ADG81752** Thermincola ADG83027** Candidatus Kuenenia CAJ71922 Planctomycetes Acetohalobium ADL11859** Firmicutes Acetohalobium ADL12609** Thermodesulfatator AEH45895 Thermodesulfobacteria Frankia ADP79978 Actinobacteria Sorangium CAN91784 δ-proteobacteria Fibrobacter ACX74327 Fibrobacteres Leptospirillum BAM07881 Nitrospirae Spirochaeta AEJ61189 Spirochaetes Treponema AAC65344 Ammonifex ACX51372** Desulfotomaculum AEG14725** Firmicutes Heliobacterium ABZ84603** Moorella ABC20154** Magnetospira CCQ73906 α-proteobacteria Nitrosococcus ADE13316 γ-proteobacteria Bacillus BAB05134 Firmicutes Brevibacillus BAH44763 Herpetosiphon ABX07093** Chloroflexi Coprothermobacter ACI16815 Firmicutes Dictyoglomus ACI18991 Dictyoglomi Thermococcus BAD85625 [23,33] Euryarchaeota Thermus BAD71725 [6] Deinococcus-Thermus Phycisphaera BAM04561 Planctomycetes Granulicella AEU36022 Acidobacteria Chthonomonas CCW35914 Armatimonadetes Fimbriimonas AIE86305 Conexibacter ADB53066 Mycobacterium CAA16116 Actinobacteria Saccharomonospora ACU97810 Chloroflexus ABY33308 Chloroflexi Herpetosiphon ABX07374** Thermococcus BAD86019 (α-galactosidase, outgroup) Euryarchaeota 0.5

Fig. 4 Maximum likelihood tree (constructed using PHYLIP [90]) fusion proteins (Fig. 1c) are in blue. Asterisks indicate that two for GH57 BEs. Proteins are indicated by the Genus name of the isoforms of GH57 BE are found in these organisms. Solid circles and source organism and GenBank accession number. Bar shows 0.5 open circles indicate nodes supported by bootstrap values of [75 and substitutions per sequence position. BEs containing loop regions [50 %, respectively. Triangles indicate that these BEs have been (shown as wavy lines in Fig. 1c) are indicated in yellow. GH57-GT4 characterized accumulation has been reported in C. limocola [68]. A gene Cyanobacteria are group of organisms that carry out annotated as BE occurs in a heterotrophic bacterium, Ig- oxygenic photosynthesis. Most of the species in this phy- navibacterium album JCM 16511 (accession number lum including Prochlorococcus marinus CCMP1986 and AFH48672), but the sequence similarity to proteins in Synechococcus elongatus PCC 7942 have one each of GH13_9 is poor. GH13_9 and GH57 BE [69]. Cyanothece sp. PCC 8802 and 123 Distribution of glucan-branching enzymes among prokaryotes 2655

Cyanothece sp. ATCC 51142 have two and three isoforms isoforms. Magnetospira sp. QH-2 is an exception among a- of GH13_9 BE, respectively, in addition to the single Proteobacteria in that it has a GH57 BE in addition to a GH57 BE [54, 56, 66]. Multiplication of BE is correlated GH13_9 BE. In b-Proteobacteria, Burkholderia multivo- with a specific trait in these species to accumulate insol- rans ATCC BAA-247 and Thiobacillus denitrificans uble, semi-crystalline a-glucan (cyanobacterial starch) ATCC 25259 have one GH13_9 BE. Azoarcus sp., Her- instead of glycogen [70, 71]. Except for the multiplication baspirillum seropedicae, Sulfuricella denitrificans skB26 of GH13_9 isoform in a limited number of species, the and Variovorax paradoxus B4 have two GH13_9 BE iso- distribution pattern of BE is uniform throughout the forms. In a BE isoform of Azoarcus sp. (CAL94413, phylum. Fig. 3), the catalytic aspartate is substituted by serine. The Deinococcus (radiation-resistant bacteria) and Thermus protein may not have the catalytic activity on its own, but (thermophilic bacteria) constitute a single phylum, but their may serve as a component of hetero-oligomeric complex BE content is distinct to each other. Deinococcus radio- with other protein(s) (e.g. with its active isoform durans has one GH13_9 BE [48] and does not possess any CAL94342), as has been reported for the complex between GH57 members. On the other hand, one GH57 BE is pre- isoamylase 1 (active) and isoamylase 2 (inactive) proteins sent in Thermus thermophilus. The protein (BAD71725) is in plants [72]. In c-Proteobacteria, a basally branched the only GH57 BE, for which enzymatic characteristics and Acidithiobacillus ferrooxidans ATCC 23270 contains one the crystal structure have been reported in Bacteria [6]. GH13_9 BE. Escherichia coli MG1655 and Vibrio cho- Distribution of BE in Firmicutes (Gram-positive bacte- lerae IEC224 also have one GH13_9 BE, and these ria with low G ? C content) shows remarkable diversity. extensively studied bacteria do not possess any member of All known types of BE are found, sometimes in combi- GH57. Moritella viscosa and Xanthomonas oryzae KACC nation, and differences are seen even between species of 10331, together with other species of Xanthomonas, have the same genus (e.g. Bacillus and Clostridium). Clostrid- two GH13_9 BEs. Methylomicrobium alcaliphilum 20Z ium clariflavum DSM 19732 and Lactococcus lactis CV56 and Nitrosococcus halophilus Nc 4 have one GH57 BE in have one GH13_8 (eukaryotic type) BE. Selenomonas addition to two GH13_9 BEs. Several species in d-Pro- ruminantium TAM6421 represents a rare example in that it teobacteria including Desulfovibrio desulfuricans ATCC has one each of GH13_8 and GH13_9 BE. Bacillus subtilis 27774 have a eukaryotic type GH13_8 BE. D. vulgaris str. 168 [50], Clostridium botulinum B str. Eklund 17B, and Miyazaki F has one GH13_9 BE, while D. africanus Lactobacillus casei BD-II have a single GH13_9 BE. Walvis Bay has two isoforms of GH13_9 BE. Sorangium Paenibacillus sp. JDR-2 and Ruminococcus obeum A2-164 cellulosum So ce56 and Myxococcus xanthus DK 1642 have two and three copies of GH13_9 BE, respectively. have one each of GH13_9 BE and GH57 BE. In e-Pro- These organisms in Firmicutes listed above do not have teobacteria, Nitratifractor salsuginis DSM 16511 and any members of GH57. Paenibacillus mucilaginosus has Sulfurimonas autotrophica DSM 16494 have a GH13_9 one each of GH13_9 and GH57 BE. Bacillus halodurans BE, as the sole member of GH13. C-125 and Clostridium acetobutylicum ATCC 824 have a In Thermotogae (mostly thermophilic bacteria), Petro- single GH57 BE, while Acetohalobium arabaticum DSM toga mobilis SJ95 has one each of GH13_9 and GH57 BE. 5501 and Desulfotomaculum kuznetsovii DSM 6115 have Thermotoga maritima MSB8 does not have GH13 BE, but two GH57 BEs. Several GH57 BEs in Firmicutes, includ- it has one GH57 family protein designated as AmyC ing one in B. halodurans, and one of two isoforms in D. (AHD18669). The protein was characterized as amylase kuznetsovii have GT4 domain fused at the C terminus to the [73] and its crystal structure was reported [74]. Based on GH57 domain, as described above (Figs. 1, 4). the amino acid sequence, AmyC is classified as a member Proteobacteria are a diverse group of Gram-negative of GH57 BE [22]. It remains to be examined whether bacteria, and most species have one or two copies of AmyC shows BE activity in addition to the hydrolase GH13_9 BEs and no GH57 BE, with several exceptions activity. across five Classes of the phylum (a- through e-Pro- Among other bacterial phyla, a single GH13_9 BE, and teobacteria). Most a-Proteobacteria do not have any no GH57 BE is found in Chlamydiae, Gemmatimonadetes, members of GH57. Species of a-Proteobacteria including Lentisphaerae, Candidate phylum NC10, Synergistetes and Paracoccus denitrificans PD1222 possess just one GH13_9 Tenericutes. Chlamydia trachomatis D/UW-3/CX (Ch- BE. Organisms in the Order Rhizobiales have two GH13_9 lamydiae) and Mycoplasma mobile 163K (Tenericutes) BEs, one each on chromosome and large plasmid. Some have one GH13_9 BE and do not possess any members of photosynthetic purple bacteria also have two copies of GH57 (many other species of Mycoplasma do not have any GH13_9 BE, but the situation varies depending on species. enzymes involved in glycogen synthesis). Gemmatimonas For example, Rhodobacter sphaeroides 2.4.1 has one aurantiaca T-27 (Gemmatimonadetes), Candidatus GH13_9 BE, while R. sphaeroides ATCC 17025 has two Methylomirabilis oxyfera (Candidate phylum NC10), and 123 2656 E. Suzuki, R. Suzuki

Thermanaerovibrio acidaminovorans DSM 6589 (Syner- one GH13_9 BE, and Methylacidiphilum infernorum V4 gistetes) have a GH13_9 BE as the sole member of GH13. has one each of GH13_9 BE and GH57 BE. One GH13_9 BE is found on the genome of Lentisphaera In contrast to the large list for Bacteria as described araneosa HTCC2155 (Lentisphaerae), although its genome above, a very limited number of BEs are known to occur in sequence remains to be completed. Archaea. Especially, no known type of BE has ever been In contrast, a single GH57 BE and no GH13 BE is identified in the phyla Crenarchaeota, as well as in present in the organisms from the phyla Armatimonadetes, Korarchaeota, Nanoarchaeota, and Thaumarchaeota. In Caldiserica, Chrysiogenetes, Defferibacteres, Dictyoglomi, Euryarchaeota, a GH13_9 BE is found in three species Elusimicrobia, Spirochaetes, and Thermodesulfobacteria. including Methanoculleus bourgensis MS2 and Picrophilus Caldisericum exile AZM16c01 (Caldiserica), Desulfu- torridus DSM 9790, as exceptions in the archaeal Domain. rispirillum indicum (Chrysiogenetes), Denitrovibrio Thermococcus kodakaraensis and other Archaea in the acetiphilus (Defferibacteres), and Thermodesulfatator Order Thermococcales have one GH57 BE. The enzyme indicus DSM 15286 (Thermodesulfobacteria) do not have a from T. kodakaraensis has been identified as the first GH57 GH13 member, while they possess a GH57 BE among BE [23] and its crystal structure has also been reported three GH57 family members. Chthonomonas calidirosea [33]. In Euryarchaeota GH57 BEs are found exclusively in T49 (Armatimonadetes), Fimbriimonas ginsengisoli Gsoil Thermococcales consisting of only three genera (Paleo- 348 (Armatimonadetes), Dictyoglomus thermophilum coccus, Pyrococcus, and Thermococcus). BE has not been (Dictyoglomi), Elusimicrobium minutum Pei191 (Elusimi- identified in the other taxa of Euryarchaeota including crobia), and organisms in Spirochaetes, Brachyspira halophiles and methanogens (other than the above men- pilosicoli WesB, Spirochaeta thermophila DSM 6578, and tioned exceptions). Despite the absence of the known types Treponema pallidum Nichols also have a GH57 BE. In BE of BE, accumulation and metabolism of glycogen have from T. pallidum (AAC65344), the catalytic residue been observed in various species of Crenarchaeota [76], aspartic acid is substituted by proline. As T. pallidum is and methanogens [77, 78]. obligate human parasite and glycogen metabolism is apparently lacking [75], the gene coding for BE may be in Correlation to the distribution of glycogen synthase the process of inactivation by mutations leading eventually to its loss. Substrate of BE, a-1,4-glucan, is produced through the Complex distribution patterns are observed in other action of glycogen synthase (GS). GS in animals and fungi bacterial phyla. Candidatus Solibacter usitatus (Aci- (EC 2.4.1.11) uses UDP-glucose as the substrate, whereas dobacteria), Aquifex aeolicus (Aquificae) [48, 57, 58], GS in prokaryotes, as well as its homolog starch synthase Fusobacterium nucleatum ATCC 25586 (Fusobacteria), (SS) in plants (EC 2.4.1.21) use ADP-glucose [2]. In terms Candidatus Nitrospira defluvii (Nitrospirae), and Plancto- of primary structure of these enzymes, animal/fungal GSs myces brasiliensis DSM 5305 (Planctomycetes) have a and prokaryotic/plant GSs (SSs) are classified into GT3 and single GH13_9 BE, while from the same phyla, Candidatus GT5, respectively [79]. The 3D structures of these glyco- Chloracidobacterium thermophilum (Acidobacteria), syltransferases show GT-B fold consisting of two b/a/b Thermovibrio ammonificans (Aquificae), Leptotrichia Rossmann-fold domains connected by a short linker [36]. buccalis C-1013-b (Fusobacteria) Leptospirillum ferrooxi- By the catalysis of GT-B fold enzymes the a-configuration dans (Nitrospirae), and Phycisphaera mikurensis NBRC of anomeric carbon in the substrate is retained, resulting in 102666 (Planctomycetes) possess one GH57 BE. One each the formation of a-1,4-glucoside linkage in GS (SS) reac- of GH13_9 and GH57 BE coexists in Fibrobacter suc- tion [35]. cinogenes (Fibrobacteres). In Chloroflexi, Thermobaculum Distribution of GS in prokaryotes is shown in Table 1. terrenum has one GH13_9 BE, Chloroflexus aurantiacus Most prokaryotes have a GT5 GS (nearly 3000 and 90 has one each of GH13_9 and GH57 BE (the catalytic members of GT5 are found in Bacteria and Archaea, glutamate residue in the GH57 BE, ABY33308, is substi- respectively, in CAZy database). In some instances, two tuted by glycine), and Herpetosiphon aurantiacus has two or even three copies of GT5 GS are found in species GH57 BEs. Due to the amino acid substitution, ABY33308 belonging to Chlorobi, Chloroflexi, Cyanobacteria, may be an inactive enzyme. The mutation may result from Deferribacteres, Firmicutes, Gemmatimonadetes, Pro- the occurrence of the redundant BEs in C. aurantiacus. teobacteria, Spirochaetes, and Verrucomicrobia. It is However, it is also possible that the inactive protein is noteworthy that GSs are found in organisms in Chlorobi, involved in the functional association with other pro- Crenarchaeota, and Euryarchaeota (methanogens), where tein(s) for glycogen metabolism. In Verrucomicrobia, no BEs have been found. It is possible that these organ- Akkermansia muciniphila ATCC BAA-835 possesses a isms have BE belonging to as-yet-unidentified protein eukaryotic type GH13_8 BE, Opitutus terrae PB90-1 has families. 123 Distribution of glucan-branching enzymes among prokaryotes 2657

In addition to the standard GS in GT5 family, GSs phosphate maltosyltransferase (GlgE, EC 2.4.99.16), and belonging to GT3 family are observed in a small number of formation of branched polysaccharide by BE (belonging to prokaryotes (110 members in CAZy database) including GH13_9) [67, 87, 88]. Among the enzymes in the pathway, the phyla Bacteroidetes, Planktomycetes, and d-Pro- TreS and GlgE have been classified into GH13_16 and teobacteria. It should be noted that in Bacteroidetes and d- GH13_3, respectively, in the CAZy database. Proteobacteria, GT3 GSs coexist with GH13_8 BE, the In addition to the intracellular a-glucans, mycobacteria combination being the same as that seen in animals and synthesize extracellular capsule glucan, structurally similar fungi. These enzymes may be acquired from Eukaryotes to glycogen, for the evasion of host immune system [66]. through lateral gene transfer (LGT). Three enzymes coexist for a-glucan elongation in Furthermore, GSs characterized in species of Acti- mycobacteria, GS, a-glucosyltransferase, and GlgE, as nobacteria, Corynebacterium glutamicum [80], described above. Studies in M. tuberculosis showed that a- Streptomyces coelicolor [81], and Mycobacterium tuber- glucosyltransferase (also responsible for the synthesis of culosis [82] are classified into GT4 family. Proteins MGLPs) and GS were involved in the production of showing high sequence similarity to GT4 GSs in Acti- glycogen and extracellular capsule glucan, respectively, nobacteria are found in Chloroflexus aurantaricus and although they seemed to have partially overlapping roles Lentisphaera araneosa, and they have been tentatively [66, 67]. While the occurrence of extracellular capsule assigned here as GT4 GS (Table 1). glucan and MGLPs is confined in mycobacteria and related Actinobacteria, enzymes in GlgE pathway are widely dis- tributed in prokaryotes, mostly in Bacteria [88]. In the Novel routes for a-glucan biosynthesis CAZy database, approximately 1000 members each of TreS and GlgE are found. In contrast to the glucan elon- Apart from the classical AGPase–GS pathway, two other gation step, the role of branch formation by BE seems to be routes of a-glucan production have been described in less redundant. The gene coding for GH13_9 BE in M. Mycobacterium tuberculosis and other Actinobacteria: (1) tuberculosis could not be inactivated, indicating its essen- synthesis of methylglucose lipopolysaccharides (MGLPs) tiality for viability [66]. In contrast, GH13_9 BE in and (2) maltose-1-phosphate maltosyltransferase (GlgE) Corynebacterium glutamicum was shown to be dispensable pathway involved in the production of glycogen and/or for viability, but required for glycogen accumulation [63]. other a-glucans (see below), starting from trehalose. MGLPs are composed of 15–20 a-1,4-linked glucoses, with numerous modifications by 6-O-methylation, 3-O- Future prospects methylation (at the non-reducing end), branching with two glucoses via b-1,3-linkages (attached at second and fourth Despite the accumulated information obtained from bio- glucose residues from the reducing end), and esterification chemical and structural studies, interrelationship between at 6-O- and 4-O-positions (the latter being only at the non- the reaction specificity and protein structure of BE remains reducing end) with acetate, propionate, isobutylate, succi- still elusive. Precise pictures for the mode of substrate nate, or octanoylate [83–85]. The reducing end of MGLPs binding at the active site, and selective mechanism for the is composed of a-glucosyl-1,2-D-glycerate, which is linked specific lengths of oligosaccharides are required. While through an a-1,6-bond to the rest of the molecule [83, 84]. GH13 family enzymes have been investigated for virtually MGLPs are postulated to assume a helical conformation any types of organism, GH57 family enzymes, including with hydrophobic cavity that can accommodate fatty acid BE, have been characterized only for thermophilic bacteria derivatives, thereby regulating lipid metabolism [83, 84]. and archaea. The GH57 BEs found in various mesophilic A GT4 a-glucosyltransferase (distinct from GS) was shown bacteria remain as unexplored frontiers for future research. to be responsible for elongation of the glucan chain in As BE has not been identified in some prokaryotic linages MGLPs [86]. A GH57 BE, whose gene lies next to that of (e.g. Chlorobi and Crenarchaeota), it would be possible that the a-glucosyltransferase in mycobacteria is thought to novel BE(s) will be discovered belonging to still unrec- catalyze the formation of the single a-1,6-linkage in ognized protein family. Such new repertoires of BE, MGLPs [83, 86], although experimental proof remains to possibly exhibiting novel catalytic properties, will bring us be obtained. new opportunities for the development of basic glyco- The GlgE pathway consists of four reaction steps, iso- science and industrial application. merization of trehalose to maltose by trehalose synthase (TreS, EC 5.4.99.16), conversion to maltose-1-phosphate Acknowledgments Studies in the authors’ laboratory were supported by maltokinase (Mak or Pep2, EC 2.7.1.175), transfer of by JSPS KAKENHI Grant Numbers 25440193 to E.S. and 15K18685 to R.S. maltosyl unit for a-glucan polymerization by maltose-1- 123 2658 E. Suzuki, R. Suzuki

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