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12.4: S-adenosyl methionine and the methyl cycle - Biology

12.4: S-adenosyl methionine and the methyl cycle - Biology


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12.4: S-adenosyl methionine and the methyl cycle

S-Adenosylmethionine Metabolism and Aging

Abstract

S-Adenosylmethionine (SAM) is the universal methyl donor with an essential role in the survival of all organisms. SAM is at the heart of ancient pathways with two other key components, sulfur and iron atoms, that go back to the days of a world without atmospheric oxygen. SAM slows down aging via proper control of synthesis, maintenance, modification, and repair of DNA, RNA, proteins, and a myriad of other components. SAM monitors the synthesis of the major antioxidant glutathione via the transsulfuration pathway, while decarboxylated SAM fuels the polyamine pathway, which is vital to coat negative backbones of macromolecules and small molecules. In this way, SAM controls the redox balance, as well as accessibility and functioning of the macromolecules, with concomitant impact on replication, transcription, translation, repair, and chromatin (re)modeling. The balance between methylation and transsulfuration is important to retain good health and live longer. SAM is also involved in transcription and translation via small RNA riboswitches. Radical SAM enzymes with SAM-dependent iron–sulfur (FeS) clusters perform difficult biochemical reactions in both microorganisms and eukaryotic cells, including the energy-generating organelles, the mitochondria and chloroplasts. Radical SAM enzymes are involved in the synthesis and use of building blocks for DNA, RNA, proteins, in the synthesis of vitamins, antibiotics, and toxins, and in the formation or removal of radicals. SAM is important for the activity of the mitochondria, in intracellular and extracellular communication, and the establishment and maintenance of the microbiome. Taken together, malfunctioning of SAM is linked to disease and premature aging.


Abstract

Protein lysine methyltransferases (PKMTs) play crucial roles in normal physiology and disease processes. Profiling PKMT targets is an important but challenging task. With cancer-relevant G9a as a target, we have demonstrated success in developing S-adenosyl- l -methionine (SAM) analogues, particularly (E)-hex-2-en-5-ynyl SAM (Hey-SAM), as cofactors for engineered G9a. Hey-SAM analogue in combination with G9a Y1154A mutant modifies the same set of substrates as their native counterparts with remarkable efficiency. (E)-Hex-2-en-5-ynylated substrates undergo smooth click reaction with an azide-based probe. This approach is thus suitable for substrate characterization of G9a and expected to further serve as a starting point to evolve other PKMTs to utilize a similar set of cofactors.


VI. Activation of the Methyl Group Acceptors: Zn Thiolates and NiFeS Clusters

The methyl group acceptors listed in Table 10.1 are amines (THF, THMPT), thiols (CoM or homocysteine), and metal ions (a NiFeS cluster). Transfer of the methyl group to THF or THMPT is simply the reverse of the methylation of cobalamin by MTHF or MTHMPT and will not be discussed here, where the focus will be on methylation of homocysteine by methionine synthase, of CoM by several of the 𠇊” components, or of the Ni center in the A-Cluster of ACS.

A. Methylation of thiol acceptors

A general theme for enzymes catalyzing alkyl transfers to thiols is that they possess a catalytic Zn site, which is considered to be important in enhancing the nucleophilicity of the thiol at neutral pH (Matthews and Goulding, 1997). The role of Zn appears to be activation of the thiol group by decreasing its pKa value, since a proton is released upon binding of homocysteine to methionine synthase (Goulding and Matthews, 1997). Although the nucleophilicity of a Zn-bound thiol is less than that of a free thiolate, the pKa of the free thiol of CoM or homocysteine is too high to allow significant amounts of the thiolate to be present, and the Zn-bound thiolate is much more nucleophilic than a thiol. Thus, metal ion-catalyzed activation poises homocysteine or CoM for nucleophilic attack on an intermediate methyl donor, such as MeCbl.

A subset of these methyltransferases, including methionine synthase, MtaA, MtbA, and MtsA share a Cys-X-His-Xn-Cys motif, where Zn coordinates to the His and two Cys residues (Gencic et al., 2001 Kr࿎r et al., 2002 Tallant et al., 2001 Zhou et al., 1999). In methioine synthase, the ligation of homocysteine directly to a Zn site was shown by XAS studies using selenohomocysteine (Peariso et al., 2001). The crystal structure of the homocysteine domain of methionine synthase reveals an (αβ)8 TIM barrel, like that of the MTHF-binding domain. Although they exhibit little sequence identify, both cobalamin-dependent (MetH) and cobalamin–independent (MetE) methionine synthases contain Zn-binding sites. The Zn site in cobalamin-independent methionine synthase contains two Cys and one His ligands, while in cobalamin-dependent methionine synthase ( Fig 10.19 ), it consists of three Cys ligands ((Evans et al., 2004 Peariso et al., 1998, 2001) and references therein). Homocysteine binds similarly to a (Cys)3Zn site within the TIM barrel in betaine–homocysteine methyltransferase (BHMT) (Evans et al., 2002) .

Homocysteine ligated to Zn within the triosephosphate isomerase (TIM) barrel of the N-terminal domain of MetH. Generated from PDB ID# 1Q8A using Chimera.

The 𠇊” components of a variety of methanogenic methyltransferases also contain catalytic Zn sites that are responsible for binding CoM or homocysteine, including the MtaA component of the methanol:CoM methyltransferase system, which is responsible for methyl transfer from methyl-Co(III) on MtaC to CoM (Gencic et al., 2001). MtsA (Tallant et al., 2001) and MtbA (Kr࿎r et al., 2002), which are MeCbl:CoM methyltransferases. Other Zn enzymes that bind thiols during their catalytic mechanism include the E.coli Ada protein (Myers et al., 1994 Wilker and Lippard, 1997), S-methylmethionine:homocysteine methyltransferase (Thanbichler et al., 1999), epoxyalkane:coenzyme M transferase (Allen et al., 1999 Ensign and Allen, 2003), and protein farnesyl transferase (Huang et al., 1997 Strickland et al., 1998).

B. Methylation of the NiFeS cluster of ACS

Acetyl-CoA Synthase (ACS), encoded by the acsB gene in M. thermoacetica, is part of a complex that catalyzes the conversion of CO2, CoA, and a methyl group to acetyl-CoA, as shown on the right hand side of Fig. 10.6 . Several reviews that focus on the structure, function, and mechanism of ACS are available (Brunold, 2004 Drennan et al., 2004 Lindahl, 2004 Ragsdale, 2006 Riordan, 2004). The other component of this complex is CODH, which is encoded by the acsA gene, and catalyzes the reduction of CO2 to CO. CODH and ACS contain internal channels that interlink to form a 70 Å channel that sequesters CO and facilitates its delivery to the ACS active site (Tan et al., 2005 Doukov et al., 2008). This channel has been identified biochemically (Maynard and Lindahl, 1999 Seravalli and Ragsdale, 2000) and by X-ray crystallography (Darnault et al., 2003 Doukov et al., 2002, 2008).

The catalytic strategy of ACS is to use a Ni active site, the A-Cluster, to form organometallic intermediates. The A-Cluster consists of a [4Fe-4S] cluster bridged to a binuclear NiNi center, which contains a Ni site (Nip) that is thiolate bridged to another Ni ion in a thiolato- and carboxamidotype N2S2 coordination environment (Darnault et al., 2003 Doukov et al., 2002 Ragsdale et al., 1985 Svetlitchnyi et al., 2004). Although the details are under discussion, the basic mechanism of ACS involves metal-centered catalysis that includes the following bioorganometallic intermediates: methyl-Ni, Ni-CO, and acetyl-Ni. The mechanism involves transfer of a methyl group from the MeCbl state of the CFeSP to a Ni center, which also binds CO and CoA then, ACS catalyzes condensation of the C𠄼 and C–S bonds to form acetyl-CoA (Ragsdale and Wood, 1985). The methyl transfer reaction could occur by either a radical or an SN2-type nucleophilic mechanism. Model studies of the reaction between methyl-Co 3+ (CH3-Co 3+ dimethylglyoximate) and a Ni 1+ macrocycle provide precedent for a methyl radical transfer (Ram and Riordan, 1995 Ram et al., 1997). A radical methyl transfer would require homolysis of the CH3𠄼o bond of the methylated CFeSP, which Martin and Finke pointed out was not favorable because reduction of CH3-Co 3+ requires redox potentials (< − 1 V) that are too low for physiological electron donors (Martin and Finke, 1990). Rapid kinetic studies and stereochemical studies using a chiral methyl donor also indicate that the transmethylation reaction involves an SN2-type nucleophilic attack of Ni on the methyl group of the methylated CFeSP (CH3𠄼o 3+ ) to generate methyl-Ni and Co 1+ (Lebertz et al., 1987 Menon and Ragsdale, 1998, 1999). Thus, it is likely that the metal-to-metal methyl transfer reaction is similar to the other B12-dependent methyltransferase reactions described above. In fact, kinetic studies indicate that the Ni (I) site on ACS is as strong a nucleophile as is the Co 1+ site in the CFeSP (Tan et al., 2003).


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