Abstract
Environments previously thought to be uninhabitable offer a tremendous wealth of unexplored microorganisms and enzymes. In this paper, we present the discovery and characterization of a novel γ-carbonic anhydrase (γ-CA) from the polyextreme Red Sea brine pool Discovery Deep (2141 m depth, 44.8°C, 26.2% salt) by single-cell genome sequencing. The extensive analysis of the selected gene helps demonstrate the potential of this culture-independent method. The enzyme was expressed in the bioengineered haloarchaeon Halobacterium sp. NRC-1 and characterized by X-ray crystallography and mutagenesis. The 2.6 Å crystal structure of the protein shows a trimeric arrangement. Within the γ-CA, several possible structural determinants responsible for the enzyme’s salt stability could be highlighted. Moreover, the amino acid composition on the protein surface and the intra- and intermolecular interactions within the protein differ significantly from those of its close homologs. To gain further insights into the catalytic residues of the γ-CA enzyme, we created a library of variants around the active site residues and successfully improved the enzyme activity by 17-fold. As several γ-CAs have been reported without measurable activity, this provides further clues as to critical residues. Our study reveals insights into the halophilic γ-CA activity and its unique adaptations. The study of the polyextremophilic carbonic anhydrase provides a basis for outlining insights into strategies for salt adaptation, yielding enzymes with industrially valuable properties, and the underlying mechanisms of protein evolution.
Highlights
Recent years have seen the discoveries of extremophiles in environments previously considered uninhabitable (Madigan and Marrs, 1997; Cavicchioli et al, 2011; Antunes et al, 2017; Jorquera et al, 2019; Merino et al, 2019)
We demonstrate that CA_D encodes a γ-carbonic anhydrase
To evaluate the potential impact of the mutations, we examined the CA_D variants for their activities expressed in Wilbur-Anderson Unit per 1 mg of protein (WAU/mg) (Wilbur and Anderson, 1948; Hou et al, 2019)
Summary
Recent years have seen the discoveries of extremophiles in environments previously considered uninhabitable (Madigan and Marrs, 1997; Cavicchioli et al, 2011; Antunes et al, 2017; Jorquera et al, 2019; Merino et al, 2019). Enzymes from extremophiles gained increasing attention because they have adapted their structure and retained their function under harsh conditions, where their mesophilic homologs are non-functional (Persidis, 1998; Akal et al, 2019). Structure of Halophilic γ-Carbonic Anhydrase proteins are attractive for biotechnological and chemical industries keen on replacing traditional catalysts with enzymes. As a result, these extremozymes provide a high stereoselectivity with fewer side reactions, and lower the burden on the environment, concomitantly accelerating reaction rates (Littlechild, 2017). Extremophilic organisms, which are naturally adapted to withstand harsh conditions, provide a perspective for optimization and rational protein-engineering approaches (Liszka et al, 2012)
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