Structural and enzymatic analyses of Anabaena heterocyst-specific alkaline invertase InvB.

Anabaena sp. PCC 7120 encodes two alkaline/neutral invertases, namely InvA and InvB. Following our recently reported InvA structure, here we report the crystal structure of the heterocyst-specific InvB. Despite sharing an overall structure similar to InvA, InvB possesses a much higher catalytic activity. Structural comparisons of the catalytic pockets reveal that Arg430 of InvB adopts a different conformation, which may facilitate the deprotonation of the catalytic residue Glu415. We propose that the higher activity may be responsible for the vital role of InvB in heterocyst development and nitrogen fixation. Furthermore, phylogenetic analysis combined with activity assays also suggests the role of this highly conserved arginine in plants and cyanobacteria, as well as some proteobacteria living in highly extreme environments.

Structural Analysis of the Catalytic Mechanism and Substrate Specificity of Anabaena Alkaline Invertase InvA Reveals a Novel Glucosidase.

Invertases catalyze the hydrolysis of sucrose to glucose and fructose, thereby playing a key role in primary metabolism and plant development. According to the optimum pH, invertases are classified into acid invertases (Ac-Invs) and alkaline/neutral invertases (A/N-Invs), which share no sequence homology. Compared with Ac-Invs that have been extensively studied, the structure and catalytic mechanism of A/N-Invs remain unknown. Here we report the crystal structures of Anabaena alkaline invertase InvA, which was proposed to be the ancestor of modern plant A/N-Invs. These structures are the first in the GH100 family. InvA exists as a hexamer in both crystal and solution. Each subunit consists of an (α/α)6 barrel core structure in addition to an insertion of three helices. A couple of structures in complex with the substrate or products enabled us to assign the subsites -1 and +1 specifically binding glucose and fructose, respectively. Structural comparison combined with enzymatic assays indicated that Asp-188 and Glu-414 are putative catalytic residues. Further analysis of the substrate binding pocket demonstrated that InvA possesses a stringent substrate specificity toward the α1,2-glycosidic bond of sucrose. Together, we suggest that InvA and homologs represent a novel family of glucosidases.

Structural insights into HetR-PatS interaction involved in cyanobacterial pattern formation.

The one-dimensional pattern of heterocyst in the model cyanobacterium Anabaena sp. PCC 7120 is coordinated by the transcription factor HetR and PatS peptide. Here we report the complex structures of HetR binding to DNA, and its hood domain (HetRHood) binding to a PatS-derived hexapeptide (PatS6) at 2.80 and 2.10 Å, respectively. The intertwined HetR dimer possesses a couple of novel HTH motifs, each of which consists of two canonical α-helices in the DNA-binding domain and an auxiliary α-helix from the flap domain of the neighboring subunit. Two PatS6 peptides bind to the lateral clefts of HetRHood, and trigger significant conformational changes of the flap domain, resulting in dissociation of the auxiliary α-helix and eventually release of HetR from the DNA major grove. These findings provide the structural insights into a prokaryotic example of Turing model.

Structure of autophagy-related protein Atg8 from the silkworm Bombyx mori.

Autophagy-related protein Atg8 is ubiquitous in all eukaryotes. It is involved in the Atg8-PE ubiquitin-like conjugation system, which is essential for autophagosome formation. The structures of Atg8 from different species are very similar and share a ubiquitin-fold domain at the C-terminus. In the 2.40 A crystal structure of Atg8 from the silkworm Bombyx mori reported here, the ubiquitin fold at the C-terminus is preceded by two additional helices at the N-terminus.