CAZyme fold architecture is conserved between disparate environments despite extreme sequence divergence

ABSTRACT Microbial carbohydrate-active enzymes (CAZymes) underpin carbon cycling across Earth’s ecosystems; however, how contrasting environments shape CAZyme diversity and structural conservation remains poorly understood. Here, we applied shotgun metagenomics to compare the carbohydrate-degradation potential of two functionally prolific but physicochemically opposed ecosystems: the alkaline-saline soda lakes of the East African Rift Valley and the anaerobic ruminant gut. From 34 metagenomes (12 soda lake and 22 rumen), we recovered 371 quality-filtered metagenome-assembled genomes, of which 84% of soda lake and 52% of rumen MAGs represented novel species. Rumen communities, dominated by Bacteroidota , Fibrobacterota , and Bacillota , exhibited significantly higher taxonomic diversity and were enriched in carbohydrate catabolism and fermentation pathways. Soda lake communities, dominated by Pseudomonadota , displayed greater evolutionary divergence (lower RED scores) and were enriched in pH homeostasis, oxidative and osmotic stress, sulfur cycling, and carbon fixation pathways. To assess whether structural conservation persists despite extreme sequence divergence, we predicted three-dimensional structures for 12 representative enzymes from six glycoside hydrolase families (GH1, GH3, GH5_11, GH9, GH10, and GH28) using AlphaFold 3. All 12 structures adopted canonical GH family folds with high confidence (pTM 0.75–0.97). These results demonstrate that environmental selection drives distinct taxonomic and functional strategies for carbon processing while preserving three-dimensional CAZyme architecture, positioning soda lake and rumen metagenomes as complementary reservoirs for bioprospecting industrially relevant enzymes. IMPORTANCE Carbohydrate-active enzymes, or CAZymes, are the molecular machines that microorganisms use to break down plant material and other complex sugars, and they underpin both the global carbon cycle and many industrial processes, from biofuel production to food, feed, and textile manufacturing. In this study, we compared the CAZyme repertoires of two microbial worlds that could hardly be more different: the alkaline, salty soda lakes of the East African Rift Valley, and the anaerobic stomachs of cattle, sheep, and goats. We show that although these communities are taxonomically distinct and their enzyme sequences have diverged dramatically, the three-dimensional shapes of their key carbohydrate-degrading enzymes remain remarkably well preserved. Soda lakes, in particular, hold a large pool of previously uncharacterised enzymes, identifying them as a promising, largely untapped source of robust biocatalysts for sustainable biotechnology and industrial applications.