Cooperative Kinetics of the Glucan Phosphatase Starch Excess4.

Glucan phosphatases are members of a functionally diverse family of dual-specificity phosphatase (DSP) enzymes. The plant glucan phosphatase Starch Excess4 (SEX4) binds and dephosphorylates glucans, contributing to processive starch degradation in the chloroplast at night. Little is known about the complex kinetics of SEX4 when acting on its complex physiologically relevant glucan substrate. Therefore, we explored the kinetics of SEX4 against both insoluble starch and soluble amylopectin glucan substrates. SEX4 displays robust activity and a unique sigmoidal kinetic response to amylopectin, characterized by a Hill coefficient of 2.77 ± 0.63, a signature feature of cooperativity. We investigated the basis for this positive kinetic cooperativity and determined that the SEX4 carbohydrate-binding module (CBM) dramatically influences the binding cooperativity and substrate transformation rates. These findings provide insights into a previously unknown but important regulatory role for SEX4 in reversible starch phosphorylation and further advances our understanding of atypical kinetic mechanisms.

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

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 (ß/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.

Domain evolution in enzymes of the neopullulanase subfamily.

Among the glycoside hydrolases (GHs) classified within the Carbohydrate-Active enZyme (CAZy) database, the α-amylase family GH13 containing ~30 different enzyme specificities and more than 37 000 sequences represents one of the largest GH families. Earlier, based on a characteristic sequence motif in their fifth conserved sequence region, the two closely related subfamilies, the so-called oligo-1,6-glucosidase and neopullulanase subfamilies, were described. Currently, the two subfamilies cover several CAZy-defined GH13 subfamilies because the α-amylase family GH13 has officially been divided into 41 subfamilies. The subfamily GH13_20 also contains, in addition to neopullulanase, cyclomaltodextrinase and maltogenic amylase. These usually possess the N-terminal starch-binding domain (SBD) classified as the carbohydrate-binding module family CBM34. The present in silico study has been focused on the neopullulanase subfamily in an effort to shed some light on the evolution of its modular arrangement. The main goal was to reveal the evolutionary relationships between the catalytic domain representing the enzyme specificity and the non-catalytic SBDs. The studied set based on the CAZy subfamily GH13_20 and family CBM34 was completed by related amylolytic enzymes, such as α-amylases, glycogen debranching enzymes and amylopullulanases. It finally consisted of 74 mostly biochemically characterized GH13 enzymes. The analysed sequences were divided into nine groups based on the presence of various carbohydrate-binding module domains (CBM20 and CBM48 in addition to CBM34). A special unique domain arrangement was revealed in the the α-amylase from Bacillus sp. AAH-31, in which the three consecutive SBDs (i.e. CBM20, CBM48 and CBM34, in that order) are present at its N-terminus.

An empirical pipeline for personalized diagnosis of Lafora disease mutations.

Lafora disease (LD) is a fatal childhood dementia characterized by progressive myoclonic epilepsy manifesting in the teenage years, rapid neurological decline, and death typically within ten years of onset. Mutations in either EPM2A, encoding the glycogen phosphatase laforin, or EPM2B, encoding the E3 ligase malin, cause LD. Whole exome sequencing has revealed many EPM2A variants associated with late-onset or slower disease progression. We established an empirical pipeline for characterizing the functional consequences of laforin missense mutations in vitro using complementary biochemical approaches. Analysis of 26 mutations revealed distinct functional classes associated with different outcomes that were supported by clinical cases. For example, F321C and G279C mutations have attenuated functional defects and are associated with slow progression. This pipeline enabled rapid characterization and classification of newly identified EPM2A mutations, providing clinicians and researchers genetic information to guide treatment of LD patients.

The unique evolution of the carbohydrate-binding module CBM20 in laforin.

Laforin catalyses glycogen dephosphorylation. Mutations in its gene result in Lafora disease, a fatal progressive myoclonus epilepsy, the hallmark being water-insoluble, hyperphosphorylated carbohydrate inclusions called Lafora bodies. Human laforin consists of an N-terminal carbohydrate-binding module (CBM) from family CBM20 and a C-terminal dual-specificity phosphatase domain. Laforin is conserved in all vertebrates, some basal metazoans and a small group of protozoans. The present in silico study defines the evolutionary relationships among the CBM20s of laforin with an emphasis on newly identified laforin orthologues. The study reveals putative laforin orthologues in Trichinella, a parasitic nematode, and identifies two sequence inserts in the CBM20 of laforin from parasitic coccidia. Finally, we identify that the putative laforin orthologues from some protozoa and algae possess more than one CBM20.

In silico identification of catalytic residues and domain fold of the family GH119 sharing the catalytic machinery with the α-amylase family GH57.

The glycoside hydrolase family 119 (GH119) contains the α-amylase from Bacillus circulans and five other hypothetical proteins. Until now, nothing has been reported on the catalytic residues and catalytic-domain fold of GH119. Based on a detailed in silico analysis involving sequence comparison in combination with BLAST searches and structural modelling, an unambiguous relationship was revealed between the families GH119 and GH57. This includes sharing the catalytic residues, i.e. Glu231 and Asp373 as catalytic nucleophile and proton donor, respectively, in the predicted catalytic (ß/α)(7)-barrel domain of GH119 B. circulans α-amylase. The GH57 and GH119 families may thus define a new CAZy clan.