Crystal Structure and Active Site Engineering of a Halophilic γ-Carbonic Anhydrase

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.

Understanding High-Salt and Cold Adaptation of a Polyextremophilic Enzyme

“…Although improvements were achieved by protein engineering, these procedures are often lengthy and expensive with non-generalizable outcomes, because increased enzyme stability mostly results from specific mutations, which usually do not obey any obvious trends or patterns [ 6 , 7 , 8 , 9 , 10 , 11 ]. Alternatively, nature provides enzymes from extremophilic microorganisms that have a unique ability to grow and thrive in extreme environments such as volcanic areas, hypersaline lakes, alkaline soda lakes, deserts, and cold oceans [ 7 , 12 , 13 , 14 , 15 , 16 , 17 , 18 ]. Since the metabolic processes and physiological functions of extremophiles are adapted to prevail under harsh conditions, enzymes from these microorganisms, called extremozymes, possess unique features enabling them to carry out reactions under extreme conditions, such as the presence of up to 5.2 M salt, various surfactants, organic solvents, elevated or low temperature, and at alkaline pH [ 14 , 19 ].…”

Bioprospecting of Novel Extremozymes From Prokaryotes—The Advent of Culture-Independent Methods

“…The second reason is mainly ought to the very limited amount of characterized extremozymes. For example, alcohol dehydrogenases and γ-carbonic anhydrase (CA_D) discovered from uncharacterized archaea collected from brine pool at the bottom of the Red Sea showed sequence homology of about 30–37% to the nearest mesophilic homologs ( Grötzinger et al, 2017 ; Akal et al, 2019 ; Vogler et al, 2020 ).…”

“…In theory, it could mean that any given protein’s structure and function could be predicted solely based on its amino acid sequence in the future. Still, today only a limited amount of algorithms have experimentally been shown to be efficient in the annotation of far distant related genes ( Grötzinger et al, 2014 ; Akal et al, 2019 ; Vogler et al, 2020 ). Reliable annotation of the entire genome of an organism that is very distantly related to described organisms is not yet available.…”

“…Further advances allowed researchers to study the genome from single cells ( Grötzinger et al, 2014 , 2017 ; Akal et al, 2019 ; Karan et al, 2019 ; Vogler et al, 2020 ). Single amplified genome (SAG) technology separates individual cells before analyzing their DNA, thus giving us information about each cell’s genome instead of bulk metagenomes.…”

A Novel Carboxylesterase Derived from a Compost Metagenome Exhibiting High Stability and Activity towards High Salinity

“…Haloadaptation of halophilic enzymes has been extensively studied by amino acid sequence and 3D structure comparison [ 15 , 16 , 17 ]. The presence of an unusually high proportion of acid residues and a drastic reduction of lysine residues on the surface of proteins play a key role in haloadaptation of enzymes [ 18 , 19 ]. Comparative analyses of halotolerant and halophilic enzymes with respect to amino acid compositions or 3D structures are rare.…”