In silico analysis of family GH77 with focus on amylomaltases from borreliae and disproportionating enzymes DPE2 from plants and bacteria

Highlights

• This in silico analysis delivers 416 4-α-glucanotransferases from the family GH77.

• Thermus aquaticus amylomaltase represents the most typical prokaryotic group.

• Borreliae produce evolutionary distinct amylomaltases with unique sequence features.

• Eukaryotic disproportionating enzymes DPE2 have counterparts also among bacteria.

Abstract

The CAZy glycoside hydrolase (GH) family GH77 is a monospecific family containing 4-α-glucanotransferases that if from prokaryotes are known as amylomaltases and if from plants including algae are known as disproportionating enzymes (DPE). The family GH77 is a member of the α-amylase clan GH-H. The main difference discriminating a GH77 4-α-glucanotransferase from the main GH13 α-amylase family members is the lack of domain C succeeding the catalytic (β/α)₈-barrel. Of more than 2400 GH77 members, bacterial amylomaltases clearly dominate with more than 2300 sequences; the rest being approximately equally represented by Archaea and Eucarya. The main goal of the present study was to deliver a detailed bioinformatics study of family GH77 (416 collected sequences) focused on amylomaltases from borreliae (containing unique sequence substitutions in functionally important positions) and plant DPE2 representatives (possessing an insert of ~ 140 residues between catalytic nucleophile and proton donor). The in silico analysis reveals that within the genus of Borrelia a gradual evolutionary transition from typical bacterial Thermus-like amylomaltases may exist to family-GH77 amylomaltase versions that currently possess progressively mutated the most important and otherwise invariantly conserved positions. With regard to plant DPE2, a large group of bacterial amylomaltases represented by the amylomaltase from Escherichia coli with a longer N-terminus was identified as a probable intermediary connection between Thermus-like and DPE2-like (existing also among bacteria) family GH77 members. The presented results concerning both groups, i.e. amylomaltases from borreliae and plant DPE2 representatives (with their bacterial counterpart), may thus indicate the direction for future experimental studies.

Introduction

The glycoside hydrolase (GH) family GH77 is a monospecific family containing 4-α-glucanotransferases (EC 2.4.1.25) defined in the well-established sequence-based classification system of all carbohydrate-active enzymes, the CAZy database [1]. This family forms the clan GH-H with the main α-amylase family GH13 and the family GH70 of glucan sucrases [2], [3], [4], [5], [6]. The enzyme 4-α-glucanotransferase is known also as amylomaltase in bacteria [7], [8], [9], [10], [11], [12], [13], [14], [15] and archaeons [16], [17] or as disproportionating enzyme (D-enzyme; DPE) in plants [18], [19], [20], [21]. Within the CAZy, the family GH77 counts almost 2300 sequenced members (1% being biochemically characterized) that originate predominantly from Bacteria (more than 2200 sequences) completed by only around 30 sequences from each Archaea and Eucarya (plants and green algae) [22].

4-α-Glucanotransferase employs the retaining reaction mechanism used in the entire clan GH-H [2] to preferably catalyze inter molecular transglycosylation, shuffling α-1,4-glucan chains by cleaving and reforming α-1,4-glycosidic bonds [3]. In fact, it catalyzes the transfer of a glucan-chain from one α-1,4-glucan to another α-1,4-glucan (or to 4-hydroxyl group of glucose) or within a single linear glucan molecule to produce a cyclic α-1,4-glucan [9], [16], [18]. Concerning the degree of polymerization, the size of the cyclic α-1,4-glucan starts from 17, which is usually much higher than α-, β- and γ-cyclodextrins produced by the family GH13 cyclodextrin glucanotransferase with 6, 7 and 8 glucose molecules, respectively [3]. Amylomaltase was also described for its ability to be used for the in vitro production of enzymatically synthesized glycogen [23]. Importantly, both the enzyme produced by cultivation of Bacillus subtilis expressing the amylomaltase gene from Thermus aquaticus as well as the unnatural enzymatically synthesized glycogen were found to be safe in food production and as a food ingredient for human consumption, respectively [24], [25]. Another interesting example was achieved with the plant DPE from adzuki beans in the enzymatical synthesis of acarviosyl maltooligosaccharides (derivatives of acarbose) that can be used as new inhibitors of glycoside hydrolases [26].

The family GH77 4-α-glucanotransferases adopt an α-amylase type (β/α)₈-barrel (TIM-barrel) as a catalytic domain, like all the clan GH-H members [2] that is, however, disrupted by more insertions between the barrel β-strands [27]. Of the insertions that form the three subdomains called B1, B2 and B3, the subdomain B1 protruding out of the barrel in the place of the loop 3 connecting the strand β3 to helix α3, corresponds to domain B typical for the whole clan [2], [27]. While the subdomain B2 is unique to family GH77, the subdomain B3 could play the role of the family GH13 domain C [27]. Note that the absence of a typical antiparallel β-sandwich domain C succeeding the catalytic TIM-barrel represents the main difference discriminating a GH77 4-α-glucanotransferase from the members of the main α-amylase family GH13 [2], [6]. The catalytic machinery consists of a triad of residues — aspartic acid, glutamic acid and aspartic acid — positioned at the barrel strands β4 (catalytic nucleophile), β5 (proton donor) and β7 (transition-state stabilizer), respectively, e.g., Asp293, Glu340 and Asp395 observed in the tertiary structure of the amylomaltase from Thermus aquaticus [27]. In addition, the structures have been published also for the enzymes from Thermus thermophilus [28] and Thermus brockianus [29]. There are two more structures determined but still not published: the one of the amylomaltase from Aquifex aeolicus (Protein Data Bank (PDB) ID: 1TZ7) and the other one of potato D-enzyme (PDB ID: 1X1N); the crystallization of Corynebacterium glutamicum amylomaltase being also reported [30]. From the sequence point of view, all the family GH77 4-α-glucanotransferases can be characterized by 4–7 conserved sequence regions [11] applicable for the entire GH-H clan [31].

It is worth mentioning that the in silico analysis of hypothetical amylomaltase from Borrelia burgdorferi revealed remarkable mutations in functional positions from CSRs [32]. The most important substitution concerned the lysine in the CSR-II that replaced otherwise invariant and functional arginine positioned two residues in front of the strand-β4 catalytic nucleophile [31], [32]. Since the arginine along with the catalytic triad belonged to the only four residues conserved invariantly throughout the α-amylase family, i.e. the clan GH-H [31], the amylomaltase identified first in the B. burgdorferi genome [33] was cloned and characterized confirming that the GH77 protein really exhibits the amylomaltase activity [11]. Currently CAZy contains several putative amylomaltases from different borreliae [22]; while some of them exhibit the remarkable mutations seen in the enzyme from B. burgdorferi, there are others possessing a typical family GH77 amylomaltase sequence [11]. This phenomenon makes the amylomaltases from borreliae an attractive subject for evolutionarily oriented studies of the family GH77.

With regard to DPE, two forms named as DPE1 and DPE2 have been identified in plants and green algae [18], [19], [20], [34], [35], [36], [37]. Whereas the DPE1 represents a typical GH77 4-α-glucanotransferase with ~ 500 amino acid residues [18], [19], [20], [26], the DPE2 contains additional domains. It consists of a GH77 catalytic TIM-barrel domain (analogous to DPE1) that is preceded by two copies of starch-binding domain (SBD) of the carbohydrate-binding module (CBM) family 20 and interrupted by an insertion of ~ 140 amino acids between the catalytic nucleophile and proton donor [38]. Although there is obviously no relationship between the insert itself and the DPE2 catalytic action, the removing of the insert resulted in the enzyme inactivation [39].

The main goal of the present study was therefore to deliver a bioinformatics analysis of the family GH77 focused on amino acid sequences of amylomaltases from borreliae that in their primary structures contain unique sequence features, i.e. natural mutations in functionally important positions. A particular attention has also been paid to DPE2, containing an insert between catalytic nucleophile and proton donor and usually possessing two copies of CBM20 at their N-terminus.