Crystal Structure of a Highly Thermostable α-Carbonic Anhydrase from Persephonella marina EX-H1

Kim, Subin; Sung, Jongmin; Yeon, Jungyoon; Choi, Seung Hun; Jin, Mi Sun

doi:10.14348/molcells.2019.0029

Cited by 12 publications

(8 citation statements)

References 60 publications

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“…Previously, these different CAs were expressed by different groups using different methods and conditions. Among them, CA3-PmaCA has been previously expressed in E. coli with N-terminal His-tag [ 25 ]. PmaCA enzyme from this organism without a signal peptide was expressed in E. coli at levels five-fold higher than that with signal peptide [ 26 ].…”

Section: Resultsmentioning

confidence: 99%

Streamlining heterologous expression of top carbonic anhydrases in Escherichia coli: bioinformatic and experimental approaches

Wei,

Lunin,

Alahuhta

et al. 2024

Microb Cell Fact

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Background Carbonic anhydrase (CA) enzymes facilitate the reversible hydration of CO2 to bicarbonate ions and protons. Identifying efficient and robust CAs and expressing them in model host cells, such as Escherichia coli, enables more efficient engineering of these enzymes for industrial CO2 capture. However, expression of CAs in E. coli is challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications. Results In this study, we streamlined the process of CA expression by selecting seven top CA candidates and used two bioinformatic tools to predict their solubility for expression in E. coli. The prediction results place these enzymes in two categories: low and high solubility. Our expression of high solubility score CAs (namely CA5-SspCA, CA6-SazCAtrunc, CA7-PabCA and CA8-PhoCA) led to significantly higher protein yields (5 to 75 mg purified protein per liter) in flask cultures, indicating a strong correlation between the solubility prediction score and protein expression yields. Furthermore, phylogenetic tree analysis demonstrated CA class-specific clustering patterns for protein solubility and production yields. Unexpectedly, we also found that the unique N-terminal, 11-amino acid segment found after the signal sequence (not present in its homologs), was essential for CA6-SazCA activity. Conclusions Overall, this work demonstrated that protein solubility prediction, phylogenetic tree analysis, and experimental validation are potent tools for identifying top CA candidates and then producing soluble, active forms of these enzymes in E. coli. The comprehensive approaches we report here should be extendable to the expression of other heterogeneous proteins in E. coli.

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Section: Resultsmentioning

confidence: 99%

Streamlining heterologous expression of top carbonic anhydrases in Escherichia coli: bioinformatic and experimental approaches

Wei,

Lunin,

Alahuhta

et al. 2024

Microb Cell Fact

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“…N-terminal disulfide bonds are a common feature in periplasmic localized α-CA's. In many of the well-characterized α-CA's, this disulfide bond is implicated in the structural stability and thermostability of the enzyme [10] , [12] , [14] , [26] . In fact, given the importance of this disulfide interaction, studies have engineered this interaction into mesophilic counterparts to enhance structural and thermostability of the engineered enzymes [27] , [28] .…”

Section: Resultsmentioning

confidence: 99%

Biochemical characterization of a psychrophilic and halotolerant α–carbonic anhydrase from a deep-sea bacterium, Photobacterium profundum

Somalinga,

Foss,

Grunden

2023

AIMSMICRO

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<abstract> Prokaryotic α–carbonic anhydrases (α-CA) are metalloenzymes that catalyze the reversible hydration of CO2 to bicarbonate and proton. We had reported the first crystal structure of a pyschrohalophilic α–CA from a deep-sea bacterium, <italic>Photobacterium profundum</italic> SS9. In this manuscript, we report the first biochemical characterization of <italic>P. profundum</italic> α–CA (PprCA) which revealed several catalytic properties that are atypical for this class of CA's. Purified PprCA exhibited maximal catalytic activity at psychrophilic temperatures with substantial decrease in activity at mesophilic and thermophilic range. Similar to other α–CA's, Ppr9A showed peak activity at alkaline pH (pH 11), although, PprCA retained 88% of its activity even at acidic pH (pH 5). Exposing PprCA to varying concentrations of oxidizing and reducing agents revealed that N-terminal cysteine residues in PprCA may play a role in the structural stability of the enzyme. Although inefficient in CO2 hydration activity under mesophilic and thermophilic temperatures, PprCA exhibited salt-dependent thermotolerance and catalytic activity under extreme halophilic conditions. Similar to other well-characterized α–CA's, PprCA is also inhibited by monovalent anions even at low concentrations. Finally, we demonstrate that PprCA accelerates CO2 biomineralization to calcium carbonate under alkaline conditions. </abstract>

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“…Sequences for previously crystallized α-CAs from S. azorense (SazCA, PDB ID: 4 × 5 s) [ 23 , 51 ], S. yellowstonense sp. YO3AOP1 (SspCA, PDB ID: 4G7A) [ 52 , 53 ], P. marina (PmCA1 and PmCA2, PDB IDs 6IM3 and 6EKI, respectively) [ 28 , 29 , 54 ] and T. ammonificans (TaCA, PDB ID:4C3T) [ 16 ] were obtained from the Protein Data Bank [ 55 ]. The sequence from C. mediatlanticus TB-2 [ 56 , 57 ] was obtained from NCBI (Accession number WP 007474387.1).…”

Section: Methodsmentioning

confidence: 99%

Section: Sequence Retrieval Analyses and Mutation Identificationmentioning

confidence: 99%

“…In post-combustion capture, the gas mixture is released at very high temperatures; thus, the process relies heavily on the thermostability properties of the CA [ 22 ]. Therefore, we determined that investigating thermostable CAs in thermophilic organisms, a practice previously recorded in the literature, was a justifiable approach [ 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. There have been previous attempts to enhance CA thermostability through protein engineering [ 30 , 31 , 32 , 33 ].…”

Section: Introductionmentioning

confidence: 99%

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Alpha Carbonic Anhydrase from Nitratiruptor tergarcus Engineered for Increased Activity and Thermostability

Manyumwa,

Zhang,

Jers

et al. 2024

IJMS

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The development of carbon capture and storage technologies has resulted in a rising interest in the use of carbonic anhydrases (CAs) for CO2 fixation at elevated temperatures. In this study, we chose to rationally engineer the α-CA (NtCA) from the thermophilic bacterium Nitratiruptor tergarcus, which has been previously suggested to be thermostable by in silico studies. Using a combination of analyses with the DEEPDDG software and available structural knowledge, we selected residues in three regions, namely, the catalytic pocket, the dimeric interface and the surface, in order to increase thermostability and CO2 hydration activity. A total of 13 specific mutations, affecting seven amino acids, were assessed. Single, double and quadruple mutants were produced in Escherichia coli and analyzed. The best-performing mutations that led to improvements in both activity and stability were D168K, a surface mutation, and R210L, a mutation in the dimeric interface. Apart from these, most mutants showed improved thermostability, with mutants R210K and N88K_R210L showing substantial improvements in activity, up to 11-fold. Molecular dynamics simulations, focusing particularly on residue fluctuations, conformational changes and hydrogen bond analysis, elucidated the structural changes imposed by the mutations. Successful engineering of NtCA provided valuable lessons for further engineering of α-CAs.

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Crystal Structure of a Highly Thermostable α-Carbonic Anhydrase from Persephonella marina EX-H1

Cited by 12 publications

References 60 publications

Streamlining heterologous expression of top carbonic anhydrases in Escherichia coli: bioinformatic and experimental approaches

Streamlining heterologous expression of top carbonic anhydrases in Escherichia coli: bioinformatic and experimental approaches

Biochemical characterization of a psychrophilic and halotolerant α–carbonic anhydrase from a deep-sea bacterium, <i>Photobacterium profundum</i>

Alpha Carbonic Anhydrase from Nitratiruptor tergarcus Engineered for Increased Activity and Thermostability

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