SAHH enzyme (overview)

Introduction

The enzyme adenosylhomocysteinase (AHCY) is widely found in various organisms, such as bacteria, nematodes, yeast, plants, insects, and vertebrates.

In mammals, AHCY plays a crucial role in the reversible conversion of S-adenosylhomocysteine (SAH) into adenosine and L-homocysteine. AHCY is part of the one-carbon metabolic cycle, a fundamental process that facilitates the transfer of one-carbon units for various biological processes like purine and thymidine synthesis, amino acid balance (cysteine, serine, and methionine), cellular redox control, and epigenetic regulation.

SAH is produced by S-adenosyl-L-methionine (SAM)-dependent methyltransferases, which transfer methyl groups from SAM to different cellular components, including nucleic acids and proteins.

Regulation

By Adenosine and Homocysteine

n thermodynamic equilibrium, the reaction is largely favored toward the synthesis of SAH in vitro, but efficient removal of adenosine and homocysteine enables the net breakup of SAH in vivo (R1)

In vitro binding experiments have shown that bovine AHCY endows two adenosine binding sites, and their usage depends on the enzyme-bound NAD+/NADH ratio (Kloor et al., 2003).

With a low affinity, adenosine binds the AHCY-NAD+ at the catalytic domain while, with high affinity, adenosine binds the enzymatically inactive AHCY-NADH at the cofactor domain (Kloor et al., 2003).

Despite the importance of NAD+ as a cofactor, whether an intracellular fluctuation in NAD+/NADH concentrations influences AHCY activity or its adenosine binding in vivo remains unknown. (R1)

Inhibition by Acetylation

Acetylation of mammalian SAHH at both Lys401 and Lys408 lowers catalytic constant 3-fold.

Proteomic analysis has revealed that mammalian AHCYs are acetylated at lysines 401 and 408 within their C-terminal tail.

In vitro, bi-acetylated-K401/408 human AHCY displays a threefold reduction of the catalytic constant and two-fold increase of SAH Km . Comparative analyses between unmodified and acetylated structures of AHCY indicate a local hydrogen-bound alteration in the vicinity of the modified lysine residues, indicating that slight structural changes in AHCY might have a significant functional impact on its catalytic activity. (R1)

Inhibition by 2-hydroxyisobutyrylation (hib) of Lys186

(R1)

Inhibition by β-hydroxybutyrylation (bhb) of several lysines (20, 43, 188, 204, 389, and 405)

In particular, forced K-bhb inhibits AHCY activity in mouse embryonic fibroblast (MEFs) and mouse liver (Koronowski et al., 2021).

Enzymatic assays from cells ectopically expressing mutants show that K188R, K389R, and K405R substitutions compromise AHCY activity (Koronowski et al., 2021). (R1)

Glycosylation

In addition to lysine modification, mouse AHCY is posttranslationally modified with an O-linked β-N-acetylglucosamine sugar (O-GlcNAcylation) at threonine 136 (Zhu et al., 2020).

The oligomerization capacity of AHCY (and therefore, its enzymatic activity) is reduced by mutation of threonine 136 to alanine (T136A), as well as by pharmacological inhibition of glycosylation (Zhu et al., 2020).

Importantly, mouse embryonic stem cells (mESCs) expressing AHCY-T136A mutant display a reduced proliferation and low alkaline phosphatase staining (Zhu et al., 2020), suggesting that AHCY glycosylation is important to balance self-renewal and pluripotency in mESCs. (R1)

AHCY undergoes T136 O-GlcNAcylation, which promotes its activity by increasing its tetrameric assembly and its affinity with homocysteine (R2)

Activation by sodium and potassium

Note: in mouse, plants and bacteria. Unclear if this is applicable to humans too.

For instance, the resolved structure of the mouse AHCY has sodium cation allocated in the C-terminal hinge region, contributing to the recognition of the substrate, similarly as the plant enzyme (Brzezinski et al., 2008).

In addition, the AHCY from Pseudomonas aeruginosa binds potassium cations, and kinetic studies indicate that potassium stimulates AHCY enzymatic activity and ligand binding (R1)

Consequences of SAHH dysfunction

1. Increased oxidative stress, due to reduction in the flux homocysteine into the transsulfuration pathway for glutathione production
2. Reduced methylation potential
3. Adenosine trap: replication stress, caused by decreased availability of adenosine for nucleotide production.

Although the mechanisms underlying increased aging and tissue degeneration in AHCY-deficient animals have not been experimentally determined, they are likely due to: (i) a reduced potential of the transmethylation reaction for specific substrates; (ii) increased oxidative stress, as a consequence of a reduction in the flux homocysteine into the transsulfuration pathway for glutathione production; and/or (iii) replication stress, caused by decreased availability of adenosine for nucleotide production. (R1)

The cytotoxic effects can be reversed by adenosine supplementation, thus suggesting that mild inactivation of AHCY may cause cellular stress in liver cells, due to adenosine depletion (R1)