Alpha-1,4-transglycosylation Activity of GH57 Glycogen Branching Enzymes Is Higher in the Absence of a Flexible Loop with a Conserved Tyrosine Residue.

Starch-like polymers can be created through the use of enzymatic modification with glycogen branching enzymes (GBEs). GBEs are categorized in the glycoside hydrolase (GH) family 13 and 57. Both GH13 and GH57 GBEs exhibit branching and hydrolytic activity. While GH13 GBEs are also capable of α-1,4-transglycosylation, it is yet unknown whether GH57 share this capability. Among the four crystal structures of GH57 GBEs that have been solved, a flexible loop with a conserved tyrosine was identified to play a role in the branching activity. However, it remains unclear whether this flexible loop is also involved in α-1,4-transglycosylation activity. We hypothesize that GH57 GBEs with the flexible loop and tyrosine are also capable of α-1,4-transglycosylation, similar to GH13 GBEs. The aim of the present study was to characterize the activity of GH57 GBEs to investigate a possible α-1,4-transglycosylation activity. Three GH57 GBEs were selected, one from Thermococcus kodakarensis with the flexible loop and two beta-strands; one from Thermotoga maritima, missing the flexible loop and beta-strands; and one from Meiothermus sp., missing the flexible loop but with the two beta-strands. The analysis of chain length distribution over time of modified maltooctadecaose, revealed, for the first time, that all three GH57 GBEs can generate chains longer than the substrate itself, showing that α-1,4-transglycosylation activity is generally present in GH57 GBEs.

The Synergistic Effect of GH13 and GH57 GBEs of Petrotoga mobilis Results in α-Glucan Molecules with a Higher Branch Density.

Glycogen is a biopolymer consisting of glycosyl units, with a linear backbone connected by α-1,4-linkages and branches attached via α-1,6-linkages. In microorganisms, glycogen synthesis involves multiple enzymes, with glycogen branching enzymes (GBEs) being vital for creating α-1,6-linkages. GBEs exist in two families: glycoside hydrolase (GH) 13 and GH57. Some organisms possess either a single GH13 or GH57 GBE, while others, such as Petrotoga mobilis, have both types of GBEs. In this study, the simultaneous use of a GH13 and GH57 GBE each from Petrotoga mobilis for α-glucan modification was investigated using a linear maltodextrin substrate with a degree of polymerization of 18 (DP18). The products from modifications by one or both GBEs in various combinations were analyzed and demonstrated a synergistic effect when both enzymes were combined, leading to a higher branch density in the glycogen structure. In this cooperative process, PmGBE13 was responsible for creating longer branches, whereas PmGBE57 hydrolyzed these branches, resulting in shorter lengths. The combined action of the two enzymes significantly increased the number of branched chains compared to when they acted individually. The results of this study therefore give insight into the role of PmGBE13 and PmGBE57 in glycogen synthesis, and show the potential use of both enzymes in a two-step modification to create an α-glucan structure with short branches at a high branch density.

Structural elements determining the transglycosylating activity of glycoside hydrolase family 57 glycogen branching enzymes.

Glycoside hydrolase family 57 glycogen branching enzymes (GH57GBE) catalyze the formation of an α-1,6 glycosidic bond between α-1,4 linked glucooliogosaccharides. As an atypical family, a limited number of GH57GBEs have been biochemically characterized so far. This study aimed at acquiring a better understanding of the GH57GBE family by a systematic sequence-based bioinformatics analysis of almost 2500 gene sequences and determining the branching activity of several native and mutant GH57GBEs. A correlation was found in a very low or even no branching activity with the absence of a flexible loop, a tyrosine at the loop tip, and two ß-strands.

GH13 Glycogen branching enzymes can adapt the substrate chain length towards their preferences via α-1,4-transglycosylation.

Glycogen branching enzymes (GBEs; 1,4-α-glucan branching enzyme; E.C. 2.4.1.18) have so far been described to be capable of both α-1,6-transglycosylation (branching) and α-1,4-hydrolytic activity. The aim of the present study was to elucidate the mode of action of three distantly related GBEs from the glycoside hydrolase family 13 by in depth analysis of the activity on a well-defined substrate. For this purpose, the GBEs from R. marinus (RmGBE), P. mobilis (PmGBE1), and B. fibrisolvens (BfGBE) were incubated with a highly pure fraction of a linear substrate of 18 anhydroglucose units. A well-known and characterized branching enzyme from E. coli (EcGBE) was also taken along. Analysis of the chain length distribution over time revealed that, next to hydrolytic and branching activity, all three GBEs were capable of generating chains longer than the substrate, clearly showing α-1,4-transglycosylation activity. Furthermore, the GBEs used those elongated chains for further branching. The sequential activity of elongation and branching enabled the GBEs to modify the substrate to a far larger extent than would have been possible with branching activity alone. Overall, the three GBEs acted ambiguous on the defined substrate. RmGBE appeared to have a strong preference towards transferring chains of nine anhydroglucose units, even during elongation, with a comparably low activity. BfGBE generated an array of elongated chains before using the chains for introducing branches while PmGBE1 exhibited a behaviour intermediate of the other two enzymes. On the basis of the mode of action revealed in this research, an updated model of the mechanism of GBEs was proposed now including the α-1,4-transglycosylation activity.

Effects of Oxygen Limitation on the Biosynthesis of Photo Pigments in the Red Microalgae Galdieria sulphuraria Strain 074G.

As a consequence of the inhibition of one of the steps in the biosynthesis of the photopigments chlorophyll and phycobilin, the red microalga Galdieria partita excretes coproporphyrinogen III in the medium when growing on glucose. No coproporphyrinogen III was found when the closely related red microalgae G. sulphuraria strain 074G was grown on glucose and excessive amounts of oxygen. When under the same conditions oxygen was limiting, coproporphyrinogen III was present in the medium. We conclude that not glucose but the amount of oxygen in the medium results in the accumulation of coproporphyrinogen III. This is explained by the inactivition of the oxygen-dependent coproporphyrinogen III oxidase that converts coproporhyrinogen III to protoporphyrinogen IX, one of the intermediate steps in the biosynthesis of chlorophyl and phycobilin.