Function of your protein
AlyC3 is an present in the cold adapted strain Psychromonas sp. C-3 which metabolizes the carbon in alginate via alginate lyase for its on growth. AlyC3 facilitates β-elimination at the glycosidic 1,4-O-linkage in alginate. AlyC3 uses tetramannuronate or polymannuronate as its substrate and converts the polymer into its substituent monomers, shorter PM chains, and 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid. Chains smaller than trimannuronate are unable to be degraded as they are too short to be positioned by the residues at opposite ends of the catalytic cavity.
Biological relevance and broader implications
The protein AlyC3 plays a role in the degradation and recycling of alginate in ocean ecosystems. Alginate composes approximately 40% of the dry mass in brown algae which acts as a primary producer and carbon sink in marine ecosytems. Alginate may be involved in the production of cytokines in plants as well as other physiological functions. It is also used as a gelling agent and viscosifier for its gel-forming properties. Alginate lyases have a variety of potential applications in the food, agriculture, and pharmaceutical industries. They may even be used to treat chronic lung infections by Pseudomonas aeruginosa. Exolytic and endolytic alginate lyases working in conjunction also have the potential to produce biofuels by breaking down alginate-rich algal cell walls into its substituent monosaccharides.
Important amino acids
The residues His127, and Tyr244 are important for the catalytic function of AlyC3 as
mutating either or both amino acids to an alanine resulted in an almost inactive enzyme.
Arg82 and Tyr190 at the two ends of the catalytic canyon are the most important for binding and
positioning the alginate substrate in AlyC3’s active site. The shown is dimannuronate complexed with a malonate ion. This fits in the on a in a β-sheet on the enzyme where H127 deprotonates the substrate and Y244 is deprotonated by the oxygen in the 1,4-O-linkage between monomers degrading the substrate.
Structural highlights
AlyC3's quaternary structure consists of two two with Cyclic – C2 symmetry. The dimerization of AlyC3 is believed to be an adapation to high salinity environments. The cavity formed by a β-sheet is the most important aspect of its tertiary structure as this is where the enzyme's substrate binds. Its is
approximately 14% helices (magenta) and 44% beta strands (yellow). Both domains each consist of 7 helices and 15 strands with one disulfide bridge. The most important for binding (R82,Y190) substrate as well as those involved in catalysis (H127,Y244) all lie in on beta strands in both domains of the protein. Positive charges in the groove as well as physical shape position the substrate in its active site with the WT having a Km value of 0.24 ± 0.05 (mg/ml). A view highlights the way the substrate fits in its active site on the surface of the enzyme. Highly charged surfaces on the protein my help prevent the loss of a hydration layer caused by high salinity in its environment.
Other important features
Two other (Q125,R78) are highly conserved and help mediate the catalytic reaction by interacting with the carboxyl group of the M+1 and activate the Cα hydrogen of M+1. Another important is His141. Its hydrophilic nature is believed to be important in stabilizing the substrate in the active site as mutating this amino acid to an alanine increases the Km value to 5.28 ± 1.11 (mg/ml) and decreases the Vmax to 6976.64 ± 852.13 (units/mg). AlyC3 shows its highest levels of activity at pH 8.0 and 20°C. It also shows peak activity in solution with approximately 0.5M NaCl where the protein exists in a dimer. As the arctic ocean is saline the salt-activation of AlyC3 is likely an adaptation to retain its quaternary structure and maintain homeostasis in its environment via alginate metabolism.
