University of Groningen
The diversity of glycogen branching enzymes in microbes
Zhang, Xuewen
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Chapter 6
Summary and Prospects
Glycogen, as the major carbon and energy reserve polymer in microorganisms and animals has been linked to a broad range of physiological processes such as cell differentiation (1), environmental survival (2,3), biofilm formation (4), and virulence (5). Impairments in glycogen synthesis or degradation in humans and animals is in most cases fatal, as for example in Pompe’s disease, Laforin dis-ease, and von Gierke’s disease (6-9). In the human pathogenic bacterium Myco-bacterium tuberculosis, the causative agent of tuberculosis killing more than one million people per year, glycogen metabolism has been connected to infection and virulence (10,11). Glycogen is a polymer of α-D-anhydroglucose, consisting of a linear backbone of α-1,4-linked anhydroglucoses with side chains attached via α-1,6-O-glycosidic linkages. The key enzymes in glycogen synthesis are: phosphotransferase, phosphoglucomutase, glucose-1-phosphate adenylyltrans-ferase, glycogenin (Eukaryotes only), glycogen synthase, and glycogen branch-ing enzyme (GBE).
GBE is a member of the α-amylase family, a large collection of the enzymes act-ing on α-glucans, includact-ing glycogen, amylose, amylopectin, and trehalose. GBEs are classified in the glycoside hydrolase families 13 (GH13) and 57 (GH57). The GH13 is a very large and diverse group of enzymes sharing an (α/β)8-barrel cat-alytic domain and catalyzing more than 20 different reactions (http://www.cazy. org). Based on protein homology, GH13 GBEs are divided into the subfamily GH13_8 (mainly Eukaryotes) and GH13_9 (mainly Prokaryotes). Family GH57 has 6 enzyme specificities; α-amylase (EC 3.2.1.1); α-galactosidase (EC 3.2.1.22); amylopullulanase (EC 3.2.1.41); cyclomaltodextrinase (EC 3.2.1.54); branching enzyme (EC 2.4.1.18); 4-α-glucanotransferase (EC 2.4.1.25), which all share an (α/β)7 catalytic barrel structure.
Although the catalytic domains of the GH13 and GH57 GBEs have a different fold they share the same double displacement catalytic mechanism, with retention of the anomeric configuration. All GBEs initiate their reaction by cleaving the α-1,4-linked linear glucan donor substrate forming a covalent glucosyl-enzyme intermediate. In the second step, the glucosyl moiety is transferred to an acceptor glucan or a water molecule to form new branches or release a free glucan chain, respectively, referred to as branching activity or hydrolytic activity. In contrast to the variety of GH13 GBEs that have been characterized (12-17), only three
GH57 GBEs have been characterized to date (18-20). This in spite of the fact that over 800 GH57 GBE gene sequences are available (21,22). This discrepancy in the number of GH57 GBEs characterized and the vast number of gene sequences available triggered me to study GH57 GBEs in more detail.
Chapter 1 introduces the current insights in the glycogen metabolism in
prokar-yotic microorganisms, the similarities and differences between family GH13 and GH57 GBEs, and the application of GBEs in starch modification. Emphasis is on the current understanding of the biological function of glycogen, the metabolic pathways involved in the synthesis of glycogen and the structural basis of the hydrolysis/branching specificity of GBEs. The differences in the reaction speci-ficity of the two families of GBEs is far from understood. Most microorganisms have one type of GBE, either a GH13 or a GH57. However, various microorgan-isms, including M. tuberculosis and Petrotoga mobilis, have the genes encoding a GH13 as well as a GH57 GBE in their genome. The simultaneous presence of a GH13 and GH57 GBE in a prokaryotic genome was found to occur widely in Nature (Chapter 1).
In contrast to GH13 GBEs, GH57 GBEs have a relatively high hydrolytic activity (20). The activity of AmyC from Thermotoga maritima, shown to be an α-amyl-ase (23,24), was characterized in detail and it was concluded that it actually is a GBE with a high hydrolytic side activity (Chapter 2). The high hydrolytic activ-ity is explained from the crystal structure, which shows that AmyC has a consid-erably shorter catalytic loop (residues 213-220) than most GH57 GBEs. This loop does not reach the acceptor side of the active site groove. In addition, one of the “gatekeeper” residues important for branching activity (19) is positioned away from the active site, thus being unable to fulfill its role as gatekeeper. Sequence analysis predicts that AmyC-like GH57 enzymes are present in species closely related to T. maritima, such as Kosmotoga pacifica, and that these enzymes are also GBEs with a relatively high hydrolytic activity.
In Chapter 1 it is described that a range of microorganisms contain both a GH13 and a GH57 GBE gene in their genome. So far, none of these GBEs have been characterized in detail. In Chapter 3 the two putative GBEs, one GH13 and one GH57, from P. mobilis are overexpressed and characterized. Both GBEs convert
amylose in branched maltodextrins with a distinct degree of branching; 13% for GH13 GBE and 8.5% for GH57 GBE. The molecular weight of the GH13 GBE product is 107 Da, while that of the GH57 GBE is 104 Da. The results from chapter 3 and those of studies on the GH13 and GH57 GBEs of M. tuberculosis together suggest that the two types of GBEs have different roles in-vivo. It is proposed that the GH13 GBE is involved in glycogen synthesis, which is supported by experimental data, while the function of GH57 GBE remains unclear. In M. tu-berculosis the GH57 GBE might be involved in lipopolysaccharide synthesis, although experimental proof is lacking. The in-vivo function of the GH57 GBE is thus still a mystery.
GBEs employ a double displacement mechanism with either water (hydrolysis) or an α-glucan (branching) as acceptor. In Chapter 4 two GH13 GBEs and two GH57 GBEs are characterized in detail to understand if and how the two family GBEs differ in activity and specificity. The GH13 GBEs have a substantially higher activity towards amylose than the GH57 GBEs. In addition, the GH57 GBEs displayed a considerable higher hydrolytic activity than GH13 GBEs. With respect to the products formed, the GH13 GBEs branched amylose or amylopec-tin more than GH57 GBEs. Moreover, the GH13 GBEs synthesized branched α-glucans with a narrow molecular weight distribution, while the GH57 GBEs products consisted of two or three molecular weight fractions. Most likely the relatively high hydrolytic activity of the GH57 GBEs is not compatible with the synthesis of branched α-glucans with a narrow molecular weight distribution. The results also indicate that the R. marinus GBE, which is commercially availa-ble as Branchzyme of Novozymes, is currently the best GBE for industrial starch processing; this enzyme only slightly hydrolyses the starch resulting in very little formation of low molecular weight material and makes a highly branched malto-dextrin (10%) with a narrow molecular weight distribution.
The GBE modified starches have several advantages over native starches, such as an increased degree of branching and solubility, smaller molecules, and lower viscosity. The higher proportion of α-1,6-linkages makes the branched maltodex-trins more resistant to degradative enzymes such as α-amylases and amylogluco-sidases, the later hydrolyzing α-1,6-linkages at a lower rate than α-1,4-linkages, and thereby makes the (highly) branched maltodextrins slowly digestible. Some
highly branched maltodextrins have been used in certain specific applications such as the formulation of peritoneal dialysis solution and sport drinks. Chapter
5 analyzes the digestibility of a series of GBE modified starches with distinct
de-gree of branching from 5 to 14%. The various highly branched maltodextrins are digested at different rates. The products with a higher branch density and shorter internal chain length contain more slowly digestible starch and more resistant starch. These results suggest that the starches treated by branching enzymes have a slowly digestible character, which could be used to control postprandial glucose levels.
In conclusion, the results in this thesis have extended the understanding of the catalytic specificity of GBEs, especially those of GH57. The GH57 GBEs display a relatively higher hydrolytic activity than GH13 GBEs. Some GH57 GBEs, such as AmyC from T. maritima, show up to 29% hydrolytic activity. In the case of AmyC, the high hydrolytic activity is correlated to the length of the “LOOP”, a stretch of 20-25 amino acids near the top of the active site. GH57 GBEs such as that of T. kodakarensis KOD1 have a long loop which almost closes the active site and positions a tyrosine residue into the active site (19,20). This tyrosine is im-portant for the branching activity of the GBE of T. thermophilus (20). In AmyC and related GBEs this loop is much shorter and does not reach the active site(23). Engineering the loop of AmyC into KOD1, and vice versa, and mutate the tyros-ine should tell us more on the role of the loop and the tyrostyros-ine.
So far it is not clear if the high hydrolytic activity observed for AmyC is an “arte-fact” of the way in which the activity is measured in-vitro. GBEs are intracellular enzymes “seeing” growing linear α-glucan chains as substrate in an environment with much less water than in the test tubes. In addition, the concentration of sub-strate in the cell is not known. All these parameters, nature and concentration of substrate, concentration of enzyme, and the amount of water all can influence the activity and specificity of the GBE. Two approaches to get some idea on the natural activity and specificity of AmyC are isolating glycogen from T. maritima and synthesizing glycogen in-vitro by combining glycogen synthase or starch/ potato phosphorylase with the GBE (17,25). Potato/starch phosphorylase, com-monly known as α-glucan phosphorylase (E.C. 2.4.1.1), catalyzes the reversible transfer of the glucose unit of glucose-1-phosphate to the non-reducing end of an
α,1-4 glucan chain with the release of phosphate (26). The Noble Prize 1947 was awarded to Cori and Cori for their instrumental work on polysaccharide phos-phorylases (27). By combining potato phosphorylase with glucose-1-phosphate, a primer such as maltoheptaose, and a GBE in various concentrations, the in-vitro glycogen synthesis can be mimicked. Van der Vlist et al. (25) have shown that using the Deinococcus geothermalis GBE a branched maltodextrin of relatively high molecular weight can be obtained. The question is whether in such an en-zyme cocktail AmyC still has a relatively high hydrolytic activity. The structure of the in-vitro synthesized branched α-glucan can subsequently be compared to that of glycogen extracted from T. maritima cells. So far, the glycogen structure of T. maritima has not been described in the scientific literature, making it not possible to compare the structure of the branched maltodextrins made from am-ylose with the natural glycogen of T. maritima.
The enzyme cocktail approach can also be used to study the GH13 and GH57 GBEs of P. mobilis and M. tuberculosis and get more insights into the roles of these two enzymes in glycogen synthesis. It is still not understood why certain microorganisms have multiple GBEs and which roles these GBEs play. For M. tuberculosis, it seems that the GH13 GBE is directly involved in glycogen syn-thesis while the GH57 GBE may play a role in the synsyn-thesis of the cell wall li-popolysaccharides (28,29). Gusthart (30) studied the M. tuberculosis GH57 GBE in detail. The protein was successfully produced in E. coli, but no activity could be detected on a range of glucans, including p-nitrophenyl-α-D-glucopyranoside, amylose, amylopectin, maltodextrin, and glycogen. What became clear from this PhD thesis is that the GH57 GBE of M. tuberculosis is not an ordinary GBE. Further research on this and the P. mobilis GH57 GBE should shed some light on the activity and role of these peculiar GBEs.
The GH57 GBE (Rv3031), the glycogen synthase (Rv3032), and the S-adenosyl-methionine-dependent methyltransferase (Rv3030) are located in the same gene cluster of M. tuberculosis, and have been proposed to synthesize lipopolysaccha-ride (28,29). However, although the GH57 GBE has been identified in P. mobilis, the other two genes are absent from the genome sequence (31), which suggests that the GH57 GBE of P. mobilis performs a different role in-vivo. The bacteria with “toga” can survive at deep marine and oil well with high osmotic stress, so
it is very likely that P. mobilis produces stress-protectant molecules to balance the external pressure. The protectants trehalose and cellobiose produced from α-glucans related genes are present in P. mobilis (32), so it seems likely that the GH57 GBE is in one way or another linked to or even involved in the synthesis of such protectants in P. mobilis. Further genetic research on P. mobilis should give some ideas on this.
New biopolymers have been created by using GBEs as a tool to increase branch-ing frequency of post-harvest starches. The highly branched products made from native starches by the action of GBEs have potential in industrial applications, such as sport drink ingredient, slowly digestible starch, and film coating (33-37). The structural properties of the branched glucans can be further diversified by combining the action of GBEs with other enzymes. For example, the amylomal-tase and GBE have been used together to produce highly branched polymers with more short side chains compared to GBE products (38); β-amylase has been applied to hydrolyse GBE products to obtain extremely branched α-glucans (39); potato phosphorylase in combination with GBE produces branched α-glucans with a high Mw (25). Following these ideas, it is proposed to use sucrose as an ideal substrate to produce highly branched α-glucans and fructose. Sucrose is a cheap, renewable biomaterial which is plentiful available while the fructose as the byproduct has more wide applications than sucrose, such as sweetener or as substrate for E. coli or yeast producing succinic acid and ethanol (40,41).
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