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Thesis
Activity and substrate specificity of cobinamide/cobamide amidohydrolase CbiZ in diverse bacteria
Master of Science (M.S.), Drexel University
Aug 2021
DOI:
https://doi.org/10.17918/00000850
Abstract
Cobamides, including vitamin B12 (cobalamin), are essential micronutrients for many organisms but are only biosynthesized by some archaea and bacteria. Cobamide variants differ in the identity of the lower base of the nucleotide loop. Many cobamide-dependent enzymes exhibit specificity for certain variants, which can impart specific biosynthetic or nutritional cobamide requirements. Some organisms can import cobamides and use them directly, while others can complete biosynthesis from imported intermediates (salvaging) or convert an imported cobamide to another variant (remodeling). One remodeling pathway involves amidohydrolase CbiZ, which cleaves the nucleotide loop from a cobamide or biosynthetic intermediate cobinamide, allowing for incorporation of a different lower base. CbiZ has only been studied in some archaea and Rhodobacter sphaeroides. Whereas archaeal CbiZ cleaves cobalamin, the Rhodobacter homolog cleaves pseudocobalamin. With the presence of dozens of other cobamides in different environments, we hypothesized that other CbiZs have different substrate specificities. With no structure or active site known, we used protein similarity networks to identify clusters of divergent CbiZ sequences. In vitro and in a heterologous expression model, we show that the CbiZs from Bacillus badius and Rossellomorea vietnamensis cleave cobinamide and the cobamides cobalamin, pseudocobalamin, and p-cresolylcobamide. We also show that two CbiZs from Sporomusa ovata demonstrate distinct cobamide specificities in a heterologous expression model. Additionally, we report that B. badius requires exogenous DMB-ribose to complete cobalamin biosynthesis and that R. vietnamensis salvages cobinamide and DMB-ribose to biosynthesize cobalamin. These results expand our understanding of CbiZ-mediated remodeling, which plays an important role in microbial communities.
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Details
- Title
- Activity and substrate specificity of cobinamide/cobamide amidohydrolase CbiZ in diverse bacteria
- Creators
- Daniel Stephen Kantner
- Contributors
- Joris Beld (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Master of Science (M.S.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- vii, 81 pages
- Resource Type
- Thesis
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology; College of Medicine; Drexel University
- Other Identifier
- 991015411089204721