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.