Štefan Janeček

Thermophilic archaeal amylolytic enzymes

Abstract The discovery of thermophilic Archaea was one of the major scientific events of the last years, especially because their enzymes are of great interest. Effectively, these later concern numerous biotechnological applications such as the enzymatic starch-hydrolysis in glucose production. In this review, we examine the different thermophilic archaeal amylolytic enzymes already characterized, with particular attention to α-amylases, the most studied enzyme of the glycoside-hydrolase family. The ecological importance of these enzymes, as well as their biochemical properties, their structure, and their evolutionary relationships are described.

Domain evolution in the alpha-amylase family.

Abstract. The available amino acid sequences of the α-amylase family (glycosyl hydrolase family 13) were searched to identify their domain B, a distinct domain that protrudes from the regular catalytic (β/α)8-barrel between the strand β3 and the helix α3. The isolated domain B sequences were inspected visually and also analyzed by Hydrophobic Cluster Analysis (HCA) to find common features. Sequence analyses and inspection of the few available three-dimensional structures suggest that the secondary structure of domain B varies with the enzyme specificity. Domain B in these different forms, however, may still have evolved from a common ancestor. The largest number of different specificities was found in the group with structural similarity to domain B from Bacillus cereus oligo-1,6-glucosidase that contains an α-helix succeeded by a three-stranded antiparallel β-sheet. These enzymes are α-glucosidase, cyclomaltodextrinase, dextran glucosidase, trehalose-6-phosphate hydrolase, neopullulanase, and a few α-amylases. Domain B of this type was observed also in some mammalian proteins involved in the transport of amino acids. These proteins show remarkable similarity with (β/α)8-barrel elements throughout the entire sequence of enzymes from the oligo-1,6-glucosidase group. The transport proteins, in turn, resemble the animal 4F2 heavy-chain cell surface antigens, for which the sequences either lack domain B or contain only parts thereof. The similarities are compiled to indicate a possible route of domain evolution in the α-amylase family.

The carbohydrate-binding module family 20--diversity, structure, and function.

Starch‐active enzymes often possess starch‐binding domains (SBDs) mediating attachment to starch granules and other high molecular weight substrates. SBDs are divided into nine carbohydrate‐binding module (CBM) families, and CBM20 is the earliest‐assigned and best characterized family. High diversity characterizes CBM20s, which occur in starch‐active glycoside hydrolase families 13, 14, 15, and 77, and enzymes involved in starch or glycogen metabolism, exemplified by the starch‐phosphorylating enzyme glucan, water dikinase 3 from Arabidopsis thaliana and the mammalian glycogen phosphatases, laforins. The clear evolutionary relatedness of CBM20s to CBM21s, CBM48s and CBM53s suggests a common clan hosting most of the known SBDs. This review surveys the diversity within the CBM20 family, and makes an evolutionary comparison with CBM21s, CBM48s and CBM53s, discussing intrafamily and interfamily relationships. Data on binding to and enzymatic activity towards soluble ligands and starch granules are summarized for wild‐type, mutant and chimeric fusion proteins involving CBM20s. Noticeably, whereas CBM20s in amylolytic enzymes confer moderate binding affinities, with dissociation constants in the low micromolar range for the starch mimic β‐cyclodextrin, recent findings indicate that CBM20s in regulatory enzymes have weaker, low millimolar affinities, presumably facilitating dynamic regulation. Structures of CBM20s, including the first example of a full‐length glucoamylase featuring both the catalytic domain and the SBD, are summarized, and distinct architectural and functional features of the two SBDs and roles of pivotal amino acids in binding are described. Finally, some applications of SBDs as affinity or immobilization tags and, recently, in biofuel and in planta bioengineering are presented.

Starch-binding domains in the post-genome era

Abstract.Starch belongs to the most abundant biopolymers on Earth. As a source of energy, starch is degraded by a large number of various amylolytic enzymes. However, only about 10% of them are capable of binding and degrading raw starch. These enzymes usually possess a distinct sequence-structural module, the so-called starchbinding domain (SBD). In general, all carbohydrate-binding modules (CBMs) have been classified into the CBM families. In this sequence-based classification the individual types of SBDs have been placed into seven CBM families: CBM20, CBM21, CBM25, CBM26, CBM34, CBM41 and CBM45. The family CBM20, known also as a classical C-terminal SBD of microbial amylases, is the most thoroughly studied. The three-dimensional structures have already been determined by X-ray crystallography or nuclear magnetic resonance for SBDs from five CBM families (20, 25, 26, 34 and 41), and the structure of the CBM21 has been modelled. Despite differences among the amino acid sequences, the fold of a distorted β-barrel seems to be conserved together with a similar way of substrate binding (mainly stacking interactions between aromatic residues and glucose rings). SBDs have recently been discovered in many non-amylolytic proteins. These may, for example, have regulatory functions in starch metabolism in plants or glycogen metabolism in mammals. SBDs have also found practical uses.

α-Amylase: an enzyme specificity found in various families of glycoside hydrolases

Abstractα-Amylase (EC 3.2.1.1) represents the best known amylolytic enzyme. It catalyzes the hydrolysis of α-1,4-glucosidic bonds in starch and related α-glucans. In general, the α-amylase is an enzyme with a broad substrate preference and product specificity. In the sequence-based classification system of all carbohydrate-active enzymes, it is one of the most frequently occurring glycoside hydrolases (GH). α-Amylase is the main representative of family GH13, but it is probably also present in the families GH57 and GH119, and possibly even in GH126. Family GH13, known generally as the main α-amylase family, forms clan GH-H together with families GH70 and GH77 that, however, contain no α-amylase. Within the family GH13, the α-amylase specificity is currently present in several subfamilies, such as GH13_1, 5, 6, 7, 15, 24, 27, 28, 36, 37, and, possibly in a few more that are not yet defined. The α-amylases classified in family GH13 employ a reaction mechanism giving retention of configuration, share 4–7 conserved sequence regions (CSRs) and catalytic machinery, and adopt the (β/α)8-barrel catalytic domain. Although the family GH57 α-amylases also employ the retaining reaction mechanism, they possess their own five CSRs and catalytic machinery, and adopt a (β/α)7-barrel fold. These family GH57 attributes are likely to be characteristic of α-amylases from the family GH119, too. With regard to family GH126, confirmation of the unambiguous presence of the α-amylase specificity may need more biochemical investigation because of an obvious, but unexpected, homology with inverting β-glucan-active hydrolases.

The evolution of starch-binding domain

Amylolytic enzymes belonging to three distinct families of glycosidases (13, 14, 15) contain the starch‐binding domain (SBD) positioned almost exclusively at the C‐terminus. Detailed analysis of all available SBD sequences from 43 different amylases revealed its independent evolutionary behaviour with regard to the catalytic domains. In the evolutionary tree based on sequence alignment of the SBDs, taxonomy is respected so that fungi and actinomycetes form their own separate parts surrounded by bacteria that are also clustered according to taxonomy. The only known N‐terminal SBD from Rhizopus oryzae glucoamylase is on the longest branch separated from all C‐terminal SBDs. The 3‐dimensional (3‐D) structures of fungal glucoamylase and bacterial CGTase SBDs are compared and used to discuss the interesting SBD evolution.

Structural and evolutionary aspects of two families of non-catalytic domains present in starch and glycogen binding proteins from microbes, plants and animals.

Starch-binding domains (SBDs) comprise distinct protein modules that bind starch, glycogen or related carbohydrates and have been classified into different families of carbohydrate-binding modules (CBMs). The present review focuses on SBDs of CBM20 and CBM48 found in amylolytic enzymes from several glycoside hydrolase (GH) families GH13, GH14, GH15, GH31, GH57 and GH77, as well as in a number of regulatory enzymes, e.g., phosphoglucan, water dikinase-3, genethonin-1, laforin, starch-excess protein-4, the β-subunit of AMP-activated protein kinase and its homologues from sucrose non-fermenting-1 protein kinase SNF1 complex, and an adaptor-regulator related to the SNF1/AMPK family, AKINβγ. CBM20s and CBM48s of amylolytic enzymes occur predominantly in the microbial world, whereas the non-amylolytic proteins containing these modules are mostly of plant and animal origin. Comparison of amino acid sequences and tertiary structures of CBM20 and CBM48 reveals the close relatedness of these SBDs and, in some cases, glycogen-binding domains (GBDs). The families CBM20 and CBM48 share both an ancestral form and the mode of starch/glycogen binding at one or two binding sites. Phylogenetic analyses demonstrate that they exhibit independent behaviour, i.e. each family forms its own part in an evolutionary tree, with enzyme specificity (protein function) being well represented within each family. The distinction between CBM20 and CBM48 families is not sharp since there are representatives in both CBM families that possess an intermediate character. These are, for example, CBM20s from hypothetical GH57 amylopullulanase (probably lacking the starch-binding site 2) and CBM48s from the GH13 pullulanase subfamily (probably lacking the starch/glycogen-binding site 1). The knowledge gained concerning the occurrence of these SBDs and GBDs through the range of taxonomy will support future experimental research.

The evolution of putative starch-binding domains.

The present bioinformatics analysis was focused on the starch‐binding domains (SBDs) and SBD‐like motifs sequentially related to carbohydrate‐binding module (CBM) families CBM20 and CBM21. Originally, these SBDs were known from microbial amylases only. At present homologous starch‐ and glycogen‐binding domains (or putative SBD sequences) have been recognised in various plant and animal proteins. The sequence comparison clearly showed that the SBD‐like sequences in genethonin‐1, starch synthase III and glucan branching enzyme should possess the real SBD function since the two tryptophans (or at least two aromatics) of the typical starch‐binding site 1 are conserved in their sequences. The same should apply also for the sequences corresponding with the so‐called KIS‐domain of plant AKINβγ protein that is a homologue of the animal AMP‐activated protein kinase (AMPK). The evolutionary tree classified the compared SBDs into three distinct groups: (i) the family CBM20 (the motifs from genethonins, laforins, starch excess 4 protein, β‐subunits of the animal AMPK and all plant and yeast homologues, and eventually from amylopullulanases); (ii) the family CBM21 (the motifs from regulatory subunits of protein phosphatase 1 together with those from starch synthase III); and (iii) the (CBM20 + CBM21)‐related group (the motifs from the pullulanase subfamily consisting of pullulanase, branching enzyme, isoamylase and maltooligosyl trehalohydrolase).

Parallel β/α-barrels of α-amylase, cyclodextrin glycosyltransferase and oligo-1,6-glucosidase versus the barrel of β-amylase: Evolutionary distance is a reflection of unrelated sequences

The structures of functionally related β/α‐barrel starch hydrolases, α‐amylase, β‐amylase, cyclodextrin glycosyltransferase and oligo‐1,6‐glucosidase, are discussed, their mutual sequence similarities being emphasized. Since these enzymes (except for β‐amylase) along with the predicted set of more than ten β/α‐barrels from the α‐amylase enzyme superfamily fulfil the criteria characteristic of the products of divergent evolution, their unrooted distance tree is presented.

Horizontal gene transfer from Eukarya to bacteria and domain shuffling: the alpha-amylase model.

Abstractα-Amylases are present in all kingdoms of the living world. Despite strong conservation of the tertiary structure, only a few amino acids are conserved in interkingdom comparisons. Animal α-amylases are characterized by several typical motifs and biochemical properties. A few cases of such α-amylases have been previously reported in some eubacterial species. We screened the bacterial genomes available in the sequence databases for new occurrences of animal-like α-amylases. Three novel cases were found, which belong to unrelated bacterial phyla: Chloroflexus aurantiacus, Microbulbifer degradans, and Thermobifida fusca. All the animal-like α-amylases in Bacteria probably result from repeated horizontal gene transfer from animals. The M. degradans genome also contains bacterial-type and plant-type α-amylases in addition to the animal-type one. Thus, this species exhibits α-amylases of animal, plant, and bacterial origins. Moreover, the similarities in the extra C-terminal domains (different from both the α-amylase domain C and the starch-binding domain), when present, also suggest interkingdom as well as intragenomic shuffling.