NMR Structure of the R-module

Abstract

In the bacterium Azotobacter vinelandii, a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1-7) has been identified. These epimerases are responsible for the epimerization of ␤-D-mannuronic acid to ␣-L-guluronic acid in alginate polymers. The epimerases consist of two types of structural modules, designated A (one or two copies) and R (one to seven copies). The structure of the catalytically active A-module from the smallest epimerase AlgE4 (consisting of AR) has been solved recently. This paper describes the NMR structure of the R-module from AlgE4 and its titration with a substrate analogue and paramagnetic thulium ions. The R-module folds into a right-handed parallel ␤-roll. The overall shape of the R-module is an elongated molecule with a positively charged patch that interacts with the substrate. Titration of the R-module with thulium indicated possible calcium binding sites in the loops formed by the nonarepeat sequences in the N-terminal part of the molecule and the importance of calcium binding for the stability of the R-module. Structure calculations showed that calcium ions can be incorporated in these loops without structural violations and changes. Based on the structure and the electrostatic surface potential of both the A-and R-module from AlgE4, a model for the appearance of the whole protein is proposed.Alginates are unbranched copolymers of 134-linked ␤-D-mannuronic acid (M) 3 and its C-5 epimer ␣-l-guluronic acid (G) (1, 2). Alginates are initially produced as a linear polymer of M-subunits only. Thereafter, single M-subunits in the polymeric chain are converted to G-subunits by enzymes called mannuronan C-5 epimerases. The mechanical and chemical properties of alginates depend on the composition and sequence of the two subunits, in particular, whether they are arranged in MM-, MG-, or GG-blocks. GG-blocks are rigid and form gels with divalent cations such as calcium. Recently, MG-blocks were suggested also to form gels with calcium (3). Poly(M) regions, however, are more flexible without the ability to form gels with cations (4, 5). The relative amounts and distribution of M and G vary extensively among different species of both brown algae (6 -8) and bacteria (9,10) Alginate is for brown algae essentially the same as cellulose is for trees and plants. The stripes of the algae are mainly G-rich, giving them the stiffness to work as a skeleton, whereas leaves are M-rich showing flexibility (11).In Azotobacter sp., alginate is used as a capsular polysaccharide (12, 13), likewise in Pseudomonas sp. (14 -16). In Azotobacter vinelandii, the composition and function of the alginates vary significantly depending on the environmental conditions.Vegetatively growing cells produce alginates that form a loose capsule structure that is easily released into the growth medium. These alginates are typically M-rich (17), but under certain conditions of environmental stress, the cells enter a resting stage designated the "cyst" stage. The cysts are surrounded by a rigid alginate...

Structural and Mutational Characterization of the Catalytic A-module of the Mannuronan C-5-epimerase AlgE4 from Azotobacter vinelandii

“…All these positively charged residues are positioned on one side of the active site cleft. The AlgE4-R-module also has a positively charged cleft on the surface of its ␤-helix (18). Connecting the C-terminal residue of the catalytic A-module to the N terminus of the R-module by a peptide bond would create one long ␤-helix protein, in which a continuous long substrate binding groove runs over the surface of the full-length AlgE4 epimerase (18), thus rationalizing the contribution of the R-module to substrate binding.…”

“…Atomic force microscopy studies have revealed that the AlgE4 A-module binds more strongly to the alginate chain than the complete enzyme, indicating that the R-module is involved in the regulation of the enzyme-substrate binding strength (17). Nevertheless, the R-module on its own is also able to bind to the alginate chain (18).…”

Disorder-to-Order Transition in the CyaA Toxin RTX Domain: Implications for Toxin Secretion

“…Similar calcium-loaded β-roll structures have been found by X-ray crystallography in the C -terminal part of other RTX proteins, such as the metalloproteases from Serratia marescens and Serratia sp. [ 8 , 11 ] and by NMR in the R-module of the Azotobacter vinelandii calcium dependent mannuronan C-5 epimerase [ 12 ]. The structure at atomic resolution of RTX proteins in the absence of calcium has not been solved thus far due to its IDP behavior, as reviewed below.…”

1H, 13C and 15N resonances of the AlgE62 subunit from Azotobacter vinelandii mannuronan C5-epimerase

“…Previously the R-module from the smallest epimerase, AlgE4 (A–R), was found to fold as an all parallel β-roll protein similar to the repeats in toxin (RTX) proteins, but with a positively charged shallow grove on the front side with the ability to bind the polyanionic alginate (Aachmann et al 2006, 2005). AlgE4 exhibit by a processive mode of action, while AlgE6 (A-R1-R2-R3) preferentially introduces GG-blocks in the alginate (Campa et al 2004, Hartmann et al 2002).…”