Construction and analyses of hybrid Azotobacter vinelandii mannuronan C-5 epimerases with new epimerization pattern characteristics

Abstract

The secreted mannuronan C-5 epimerases from Azotobacter vinelandii form a family of seven homologous modular type enzymes, which appear to have evolved through duplications and point mutations in the individual modules. The catalytic A modules of these enzymes are responsible for generating the characteristic sequence distribution patterns of G residues in the industrially important polymer alginate by epimerizing M (beta-D-mannuronic acid) moieties to G (alpha-L-guluronic acid). Forty-six different hybrid enzymes were constructed by exchanging parts of the sequences encoding the A modules of AlgE2 (generates consecutive stretches of G residues) and AlgE4 (generates alternating structures). These hybrid enzymes introduce a variety of new monomer-sequence patterns into their substrates, and some regions important for the subsite specificity or processivity of the enzymes were identified. By using time-resolved NMR spectroscopy, it became clear that the rates for introducing alternating structures and consecutive stretches of G residues are different for each enzyme, and that it is the ratio between these rates that determines the overall epimerization pattern. These findings open up new possibilities in biotechnology and in studies of the many biological functions of alginates.

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

“…If the polymer chain has to slide through these positions it is also clear that, because of steric hindrance, O-2 and O-3 acetylated polymers cannot be epimerized, which explains why AlgE4A has no activity on such substrates. The importance of the second clamp is supported by hybrid enzyme studies in which residues 215 to 262 from AlgE4A, a processive enzyme, were transferred to AlgE2, a non-processive enzyme, converting it into a presumably processive enzyme (51).…”

Bacterial alginates: from biosynthesis to applications

“…This extensive variability might be biologically significant for the formation of the resting-stage, designated cyst, which is characteristic for the genus Azotobacter (Campos et al 1996;Høidal et al 2000). Expression of hybrid epimerase enzymes in E. coli demonstrated even further biotechnological potential, since the epimerization pattern of these hybrid enzymes were different from those of their parent enzymes (Bjerkan et al 2004). The guluronic acid content of the polymer corresponds to more interaction areas with divalent cations (especially Ca 2+ ), resulting in more rigid and gel-like polymer structures.…”

“…1999; Valla et al 2001;Bjerkan et al 2004) and the genes encoding these extracellular epimerases are not located within the alginate biosynthesis gene cluster. Recently, six different C5-epimerase encoding genes have been identified in the genome of Laminaria digitata (Nyvall et al 2003).…”

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“…G blocks are of great biological and biotechnological significance as they are a prerequisite for the formation of strong polymer gels in the presence of divalent cations like Ca2+; however the composition of the commercially available alginates vary, depending on the source of the polymer. Hence extensive work has been carried out in order to investigate the epimerization properties for each A. vinelandii C-5 epimerase, with the aim of increasing the G content of commercially available alginates, containing a wide range of initial G residues, or in order to produce alginates with specialized properties [50,52,53]. While algE4 activity introduces alternating M and G residues into substrate, the remaining six enzymes introduce a mixture of continuous stretches of G residues and alternating sequences.…”