Characterization of a novel type of carbonic anhydrase that acts without metal cofactors

Abstract Background Carbonic anhydrases (CAs) are universal metalloenzymes that catalyze the reversible conversion of carbon dioxide (CO2) and bicarbonate (HCO3-). They are involved in various biological processes, including pH control, respiration, and photosynthesis. To date, eight evolutionarily unrelated classes of CA families (α, β, γ, δ, ζ, η, θ, and ι) have been identified. All are characterized by an active site accommodating the binding of a metal cofactor, which is assumed to play a central role in catalysis. This feature is thought to be the result of convergent evolution. Results Here, we report that a previously uncharacterized protein group, named “COG4337,” constitutes metal-independent CAs from the newly discovered ι-class. Genes coding for COG4337 proteins are found in various bacteria and photosynthetic eukaryotic algae. Biochemical assays demonstrated that recombinant COG4337 proteins from a cyanobacterium (Anabaena sp. PCC7120) and a chlorarachniophyte alga (Bigelowiella natans) accelerated CO2 hydration. Unexpectedly, these proteins exhibited their activity under metal-free conditions. Based on X-ray crystallography and point mutation analysis, we identified a metal-free active site within the cone-shaped α+β barrel structure. Furthermore, subcellular localization experiments revealed that COG4337 proteins are targeted into plastids and mitochondria of B. natans, implicating their involvement in CO2 metabolism in these organelles. Conclusions COG4337 proteins shared a short sequence motif and overall structure with ι-class CAs, whereas they were characterized by metal independence, unlike any known CAs. Therefore, COG4337 proteins could be treated as a variant type of ι-class CAs. Our findings suggested that this novel type of ι-CAs can function even in metal-poor environments (e.g., the open ocean) without competition with other metalloproteins for trace metals. Considering the widespread prevalence of ι-CAs across microalgae, this class of CAs may play a role in the global carbon cycle.

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Structural insights into the LCIB protein family reveals a new group of β-carbonic anhydrases

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1616294113 ◽

2016 ◽

Vol 113 (51) ◽

pp. 14716-14721 ◽

Cited By ~ 27

Author(s):

Shengyang Jin ◽

Jian Sun ◽

Tobias Wunder ◽

Desong Tang ◽

Asaph B. Cousins ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

Active Site ◽

Phaeodactylum Tricornutum ◽

Protein Family ◽

Carbonic Anhydrases ◽

Uncharacterized Protein ◽

C Protein ◽

Functional Homolog ◽

Slow Kinetics ◽

Structural Insights

Aquatic microalgae have evolved diverse CO2-concentrating mechanisms (CCMs) to saturate the carboxylase with its substrate, to compensate for the slow kinetics and competing oxygenation reaction of the key photosynthetic CO2-fixing enzyme rubisco. The limiting CO2-inducible B protein (LCIB) is known to be essential for CCM function inChlamydomonas reinhardtii. To assign a function to this previously uncharacterized protein family, we purified and characterized a phylogenetically diverse set of LCIB homologs. Three of the six homologs are functional carbonic anhydrases (CAs). We determined the crystal structures of LCIB and limiting CO2-inducible C protein (LCIC) fromC. reinhardtiiand a CA-functional homolog fromPhaeodactylum tricornutum, all of which harbor motifs bearing close resemblance to the active site of canonical β-CAs. Our results identify the LCIB family as a previously unidentified group of β-CAs, and provide a biochemical foundation for their function in the microalgal CCMs.

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Watching a double strand break repair polymerase insert a pro-mutagenic oxidized nucleotide

Nature Communications ◽

10.1038/s41467-021-21354-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Joonas A. Jamsen ◽

Akira Sassa ◽

David D. Shock ◽

William A. Beard ◽

Samuel H. Wilson

Keyword(s):

Active Site ◽

Dna Polymerases ◽

Time Lapse ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Dsb Repair ◽

X Ray Crystallography ◽

Polymerase Fidelity ◽

Break Repair

AbstractOxidized dGTP (8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease. How DNA polymerases discriminate against oxidized and undamaged nucleotides, especially in error-prone double strand break (DSB) repair, is poorly understood. High-resolution time-lapse X-ray crystallography snapshots of DSB repair polymerase μ undergoing DNA synthesis reveal that a third active site metal promotes insertion of oxidized and undamaged dGTP in the canonical anti-conformation opposite template cytosine. The product metal bridged O8 with product oxygens, and was not observed in the syn-conformation opposite template adenine (At). Rotation of At into the syn-conformation enabled undamaged dGTP misinsertion. Exploiting metal and substrate dynamics in a rigid active site allows 8-oxodGTP to circumvent polymerase fidelity safeguards to promote pro-mutagenic double strand break repair.

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Crystal Structure of Escherichia coli Agmatinase: Catalytic Mechanism and Residues Relevant for Substrate Specificity

International Journal of Molecular Sciences ◽

10.3390/ijms22094769 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4769

Author(s):

Pablo Maturana ◽

María S. Orellana ◽

Sixto M. Herrera ◽

Ignacio Martínez ◽

Maximiliano Figueroa ◽

...

Keyword(s):

Escherichia Coli ◽

Active Site ◽

Catalytic Mechanism ◽

Conformational Dynamics ◽

High Specificity ◽

Arginine Decarboxylase ◽

Space Groups ◽

X Ray Crystallography ◽

High Dynamics ◽

Dynamics Simulations

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer’s, Parkinson’s, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.

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The Electronic Structure of FeV-cofactor in Vanadium-Dependent Nitrogenase

Chemical Science ◽

10.1039/d0sc06561g ◽

2021 ◽

Author(s):

Zhi-yong Yang ◽

Emilio Jimenez-Vicente ◽

Hayden Kallas ◽

Dmitriy A Lukoyanov ◽

Hao Yang ◽

...

Keyword(s):

Electronic Structure ◽

Active Site ◽

Resting State ◽

Metal Cofactor ◽

Open Question

The electronic structure of the active-site metal cofactor (FeV-cofactor) of resting-state V-dependent nitrogenase has been an open question, with earlier studies indicating that it exhibits a broad S = 3/2...

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Binding of Phosphate and pyrophosphate ions at the active site of human angiogenin as revealed by X-ray crystallography

Protein Science ◽

10.1110/ps.13601 ◽

2001 ◽

Vol 10 (8) ◽

pp. 1669-1676 ◽

Cited By ~ 14

Author(s):

Demetres D. Leonidas ◽

Gayatri B. Chavali ◽

Anwar M. Jardine ◽

Songlin Li ◽

Robert Shapiro ◽

...

Keyword(s):

Active Site ◽

X Ray ◽

X Ray Crystallography

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The Geometry of the Catalytic Active Site in [FeFe]-Hydrogenases is Determined by Hydrogen Bonding and Proton Transfer

10.26434/chemrxiv.7756214 ◽

2019 ◽

Author(s):

Jifu Duan ◽

Stefan Mebs ◽

Moritz Senger ◽

Konstantin Laun ◽

Florian Wittkamp ◽

...

Keyword(s):

Hydrogen Bonding ◽

Proton Transfer ◽

Active Site ◽

Time Resolved ◽

X Ray Crystallography ◽

Hydrogen Bonding Interactions ◽

Catalytic Active Site ◽

Catalytic Intermediates ◽

Time Resolved Infrared Spectroscopy

The H2 conversion and CO inhibition reactivity of nine [FeFe]-hydrogenase constructs with semi-artificial cofactors was studied by in situ and time-resolved infrared spectroscopy, X-ray crystallography, and theoretical methods. Impaired hydrogen turnover and proton transfer as well as characteristic CO inhibition/ reactivation kinetics are assigned to varying degrees of hydrogen-bonding interactions at the active site. We show that the probability to adopt catalytic intermediates is modulated by intramolecular and protein-cofactor interactions that govern structural dynamics at the active site of [FeFe]-hydrogenases.<br>

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Mechanism of IPPase shown by high resolution Neutron and X-ray crystallography

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314087889 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1211-C1211

Author(s):

Joseph Ng ◽

Ronny Hughes ◽

Michelle Morris ◽

Leighton Coates ◽

Matthew Blakeley ◽

...

Keyword(s):

High Resolution ◽

Active Site ◽

Inorganic Pyrophosphatase ◽

Inorganic Pyrophosphate ◽

X Ray ◽

X Ray Crystallography ◽

Cellular Processes ◽

Crystallographic Structures ◽

Hydrolysis Of ◽

Low Energy Conformations

Soluble inorganic pyrophosphatase (IPPase) catalyzes the hydrolysis of inorganic pyrophosphate (PPi) to form orthophosphate (Pi). The action of this enzyme shifts the overall equilibrium in favor of synthesis during a number of ATP-dependent cellular processes such as in the polymerization of nucleic acids, production of coenzymes and proteins and sulfate assimilation pathways. Two Neutron crystallographic (2.10-2.50Å) and five high-resolution X-ray (0.99Å-1.92Å) structures of the archaeal IPPase from Thermococcus thioreducens have been determined under both cryo and room temperatures. The structures determined include the recombinant IPPase bound to Mg+2, Ca+2, Br-, SO2-2 or PO4-2 involving those with non-hydrolyzed and hydrolyzed pyrophosphate complexes. All the crystallographic structures provide snapshots of the active site corresponding to different stages of the hydrolysis of inorganic pyrophosphate. As a result, a structure-based model of IPPase catalysis is devised showing the enzyme's low-energy conformations, hydration states, movements and nucleophile generation within the active site.

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