S-adenosyl-L-homocysteine hydrolase from a hyperthermophile (Thermotoga maritima) is expressed in Escherichia coli in inactive form – Biochemical and structural studies

Brzeziński, Krzysztof; Czyrko, Justyna; Sliwiak, Joanna; Nalewajko-Sieliwoniuk, Edyta; Jaskólski, Mariusz; Nocek, B.; Dauter, Zbigniew

doi:10.1016/j.ijbiomac.2017.06.065

Cited by 9 publications

(14 citation statements)

References 46 publications

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“…With regard to the occurrence of Ado2 in general, a somewhat similar situation has been observed in the structure of SAHase from Thermotoga maritima (TmSAHase; PDB entry 5tow; Brzezinski et al, 2017 ), where the cofactor binding sites of two subunits, forming one intimate dimer, are also occupied by Ado molecules ( Figure 5C ), while the remaining two subunits of the tetrameric protein are occupied by the reduced cofactor (NADH). In that case, Ado seems to be even capable of successfully competing with the cofactor for the nucleotide binding site.…”

Section: Discussionsupporting

confidence: 64%

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S-Adenosyl-L-Homocysteine Hydrolase Inhibition by a Synthetic Nicotinamide Cofactor Biomimetic

et al. 2018

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S-adenosyl-L-homocysteine (SAH) hydrolases (SAHases) are involved in the regulation of methylation reactions in many organisms and are thus crucial for numerous cellular functions. Consequently, their dysregulation is associated with severe health problems. The SAHase-catalyzed reaction is reversible and both directions depend on the redox activity of nicotinamide adenine dinucleotide (NAD+) as a cofactor. Therefore, nicotinamide cofactor biomimetics (NCB) are a promising tool to modulate SAHase activity. In the present in vitro study, we investigated 10 synthetic truncated NAD+ analogs against a SAHase from the root-nodulating bacterium Bradyrhizobium elkanii. Among this set of analogs, one was identified to inhibit the SAHase in both directions. Isothermal titration calorimetry (ITC) and crystallography experiments suggest that the inhibitory effect is not mediated by a direct interaction with the protein. Neither the apo-enzyme (i.e., deprived of the natural cofactor), nor the holo-enzyme (i.e., in the NAD+-bound state) were found to bind the inhibitor. Yet, enzyme kinetics point to a non-competitive inhibition mechanism, where the inhibitor acts on both, the enzyme and enzyme-SAH complex. Based on our experimental results, we hypothesize that the NCB inhibits the enzyme via oxidation of the enzyme-bound NADH, which may be accessible through an open molecular gate, leaving the enzyme stalled in a configuration with oxidized cofactor, where the reaction intermediate can be neither converted nor released. Since the reaction mechanism of SAHase is quite uncommon, this kind of inhibition could be a viable pharmacological route, with a low risk of off-target effects. The NCB presented in this work could be used as a template for the development of more potent SAHase inhibitors.

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Section: Discussionsupporting

confidence: 64%

“…Colors/hydrogen bonds as in A . (C) Ado2 (ball-and-stick) bound in the cofactor binding site of TmSAHase (PDB entry 5tow; Brzezinski et al, 2017 ). Lys231, equivalent of Cys289 in the BeSAHase structure, is shown in ball-and-stick representation.…”

Section: Resultsmentioning

confidence: 99%

S-Adenosyl-L-Homocysteine Hydrolase Inhibition by a Synthetic Nicotinamide Cofactor Biomimetic

et al. 2018

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“…The smaller C-terminal dimerization domain extends to the adjacent domain and stabilizes the homodimer through numerous interactions with the macromolecular environment and the cofactor molecule in all SAHases of eukaryotic origin, as well as in numerous bacterial SAHases. A different situation is observed in archaeal-type SAHase from hyperthermophilic bacterium T. maritima, where the DD is still involved in homodimer stabilization but is too short to be involved in any interactions with the cofactor molecule bound in a neighboring subunit [27,28]. However, the homodimer is a rare active form of SAHase (Figure 1a), restricted to the plant enzyme (Lupinus luteus) [29,30].…”

Section: Introductionmentioning

confidence: 93%

“…Crystallographic studies were also conducted for some bacterial SAHases, including enzymes from Mycobacterium tuberculosis [22], Bradyrhizobium elkanii [23,24], Cytophaga hutchinsonii [25], Pseudomonas aeruginosa [26], Burkholderia pseudomalei (3D64, 3GLQ, unpublished), Brucella abortus (3N58, unpublished), and Elizabethkingia anopheles (6APH, unpublished). Additionally, crystal structures of an archaeal-type SAHase from hyperthermophilic bacterium Thermotoga maritima were determined for the enzyme in its active and inactive conformations [27,28].…”

Section: Introductionmentioning

confidence: 99%

S-adenosyl-l-homocysteine Hydrolase: A Structural Perspective on the Enzyme with Two Rossmann-Fold Domains

Brzeziński

2020

Biomolecules

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S-adenosyl-l-homocysteine hydrolase (SAHase) is a major regulator of cellular methylation reactions that occur in eukaryotic and prokaryotic organisms. SAHase activity is also a significant source of l-homocysteine and adenosine, two compounds involved in numerous vital, as well as pathological processes. Therefore, apart from cellular methylation, the enzyme may also influence other processes important for the physiology of particular organisms. Herein, presented is the structural characterization and comparison of SAHases of eukaryotic and prokaryotic origin, with an emphasis on the two principal domains of SAHase subunit based on the Rossmann motif. The first domain is involved in the binding of a substrate, e.g., S-adenosyl-l-homocysteine or adenosine and the second domain binds the NAD+ cofactor. Despite their structural similarity, the molecular interactions between an adenosine-based ligand molecule and macromolecular environment are different in each domain. As a consequence, significant differences in the conformation of d-ribofuranose rings of nucleoside and nucleotide ligands, especially those attached to adenosine moiety, are observed. On the other hand, the chemical nature of adenine ring recognition, as well as an orientation of the adenine ring around the N-glycosidic bond are of high similarity for the ligands bound in the substrate- and cofactor-binding domains.

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“…SAHases usually function as 222-symmetric homotetramers, [4][5][6][7][8][9][10][11] or sometimes as homodimers. [3,12] Each subunit of the enzyme contains a tightly but noncovalently bound nicotinamide adenine dinucleotide cofactor in its oxidized form, NAD + .…”

Section: Introductionmentioning

confidence: 99%

Crystal Structure of S-adenosyl-L-homocysteine Hydrolase from Cytophaga hutchinsonii, a Case of Combination of Crystallographic and Non-crystallographic Symmetry

Czyrko¹,

Jaskólski²,

Brzeziński³

2018

Croat. Chem. Acta

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The majority of living organisms utilize S-adenosyl-L-homocysteine hydrolase (SAHase) as a key regulator of cellular methylation reactions. The unusual evolution history of SAHase genes is reflected in the phylogeny of these proteins, which are grouped into two major domains: mainly archaeal and eukaryotic/bacterial. Such a phylogeny is in contradiction to the three-domain topology of the tree of life, commonly based on 16S rRNA sequences. Within the latter domain, SAHases are classified as eukaryotic-only or bacterial-only clades depending on their origin and sequence peculiarities. A rare exception in this classification is SAHase from a cellulose-utilizing soil bacterium Cytophaga hutchinsonii (ChSAHase), as the phylogenetic analyses indicate that ChSAHase belongs to the animal clade. Here, the P21212 crystal structure of recombinant ChSAHase in ternary complex with the oxidized form of the NAD + cofactor and a reaction product/substrate (adenosine) is presented. Additionally, a sodium cation was identified in close proximity of the active site. The crystal contains two translational NCS-related intimate dimers of ChSAHase subunits in the asymmetric unit. Two complete tetrameric enzyme molecules are generated from these dimers within the crystal lattice through the operation of crystallographic twofold axes in the z direction.

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S-adenosyl-L-homocysteine hydrolase from a hyperthermophile (Thermotoga maritima) is expressed in Escherichia coli in inactive form – Biochemical and structural studies

Cited by 9 publications

References 46 publications

S-Adenosyl-L-Homocysteine Hydrolase Inhibition by a Synthetic Nicotinamide Cofactor Biomimetic

S-Adenosyl-L-Homocysteine Hydrolase Inhibition by a Synthetic Nicotinamide Cofactor Biomimetic

S-adenosyl-l-homocysteine Hydrolase: A Structural Perspective on the Enzyme with Two Rossmann-Fold Domains

Crystal Structure of S-adenosyl-L-homocysteine Hydrolase from Cytophaga hutchinsonii, a Case of Combination of Crystallographic and Non-crystallographic Symmetry

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