Diversity of the reaction mechanisms of SAM-dependent enzymes

Abstract

S-adenosylmethionine (SAM) is ubiquitous in living organisms and is of great significance in metabolism as a cofactor of various enzymes. Methyltransferases (MTases), a major group of SAM-dependent enzymes, catalyze methyl transfer from SAM to C, O, N, and S atoms in small-molecule secondary metabolites and macromolecules, including proteins and nucleic acids. MTases have long been a hot topic in biomedical research because of their crucial role in epigenetic regulation of macromolecules and biosynthesis of natural products with prolific pharmacological moieties. However, another group of SAM-dependent enzymes, sharing similar core domains with MTases, can catalyze nonmethylation reactions and have multiple functions. Herein, we mainly describe the nonmethylation reactions of SAM-dependent enzymes in biosynthesis. First, we compare the structural and mechanistic similarities and distinctions between SAM-dependent MTases and the non-methylating SAM-dependent enzymes. Second, we summarize the reactions catalyzed by these enzymes and explore the mechanisms. Finally, we discuss the structural conservation and catalytical diversity of class I-like non-methylating SAM-dependent enzymes and propose a possibility in enzymes evolution, suggesting future perspectives for enzyme-mediated chemistry and biotechnology, which will help the development of new methods for drug synthesis.

Keywords: Biocatalysis; Catalytic mechanism; Methyltransferase; Nonmethylation reaction; SAM-dependent enzyme.

Graphical abstract

Figure 1

The five classes of SAM-dependent MTases. The core domain and primary architecture of the representative enzyme in each case are shown in cartoons (top) and topology diagrams (bottom). (A) Class I MTases, exemplified by COMT (PDB: 1VID): the Rossmann fold with a seven-stranded β-sheet and flanking α-helices in the C-terminus is defined as the core domain in this class. The auxiliary N-terminus (colored in grayish-white) is specific for substrate recognition and polymerization. (B) Class II MTases, exemplified by MetH (PDB: 1MSK): the key characteristic of this class is a long antiparallel β-sheet flanked by groups of helices. (C) Class III MTases, exemplified by CbiF (PDB: 1CBF): two αβα-domains formed in the N- and C-termini anchor the active site, providing a sufficiently large pocket for large substrates and SAM. (D) Class IV MTases, exemplified by YibK (PDB: 1MXI): the partial Rossmann fold is similar to that of class I MTases but differs in the knot-like C-terminus (colored in cyan), which tucks back and inserts itself into the space between a α-helix and a β-strand. (E) Class V MTases, exemplified by Set7/9 (PDB: 1O9S): the structure is mainly composed of a series of β-strands with several flanking α-helices. The C-terminus tucks back to form a “knot” (colored in cyan). In both the cartoon and topology diagrams, α-helices and β-strands in the characteristic domain are colored orange and blue, knot-like domains are colored cyan, variant domains are colored grayish-white, and SAM molecules are colored pink.

Figure 2

Overall structure, close-up view and topology diagram of a representative radical SAM enzyme (HemN, PDB: 1OLT) are shown in a cartoon and a topology diagram. The core TIM barrel is defined as the region from the N-terminus of the strand (β1) leading to the cluster-binding loop to the C-terminus of the sixth strand (β6). In the representative enzyme HemN, the core TIM barrel containing residues from Leu53 to Asn241 is highlighted. (A) Overview of HemN. The partial (α/β)₆TIM barrel is conserved in the majority of radical SAM enzymes. The special α4a protrudes from the partial TIM barrel, and its C-terminus is oriented toward a cluster-binding loop (between β1 and α1). (B) Close-up view of the [4Fe-4S] cluster and SAM binding mode. Three cysteines (Cys62, Cys66 and Cys69 in HemN) on the loop following the first main strand in the TIM barrel are conserved for cluster binding and coordinating with three Fe ions (colored in magenta), which is referred to as the cysteine-rich motif. The amide nitrogen and carboxylate oxygen of SAM coordinate to the fourth Fe ion. The SAM adjacent to the cluster binds at the top of the TIM barrel, and the glycine-rich motif (Gly112, Gly113 and Thr114 in HemN) is defined in many radical SAM enzymes, but other SAM-binding motifs are difficult to identify on account of the large overlap of the SAM-binding regions in different enzymes. Here, we labeled both hydrophilic and hydrophobic residues of one SAM-binding region in HemN. The second SAM molecule is present in some cases, such as HemN, BioB and LipA, but the binding mode is not discussed here. (C) Topology diagram of HemN. In both the cartoon and topology diagram, α-helices and β-strands in the TIM barrel are colored orange and blue, variant domains are colored grayish-white, SAM is colored pink, Fe ions in the cluster are colored magenta, S atoms are colored yellow, residues are colored cyan, and hydrogen bonds are labeled with red dashed lines.

Figure 3

Mechanism of S_N2 or S_N2-like methylation. (A) General reaction performed by SAM-dependent MTases. Positively charged SAM acts as a methyl donor and provides a methyl group. The electron-sufficient atom attacks the methyl group, initiating S_N2-like replacement and inducing cleavage of the C–S bond. Finally, SAH is released during the production of a methylated substrate. (B) The “proximity and desolvation” mechanism, exemplified by DnrK. Methylated oxygen is in proximity to the sulfonium group of SAM at a distance of approximately 4.3 Å (distance shown in yellow). Asn256 is supposed to assist substrate stabilization. Tyr142 is adjacent to a reactive oxygen species (O-4, indicated by the black arrow) but does not have a substantial effect on the catalytic rate, indicating that DnrK catalyzes methylation through a “proximity and desolvation” mechanism. (C) The general acid/base-mediated catalytic mechanism, exemplified by NirE. Arg111 acts as a general base to deprotonate the hydrogen on C-20 of substrate, driving the S_N2-methyl transfer from SAM to C-2. Glu114 is important for correction of the orientation of Arg111. The present structure may not reflect the real position in the native enzyme because of the absence of SAM, resulting in a relatively large distance between the C-2 and S atoms in SAH (S-adenosylhomocysteine). Further details about the substrate conformation and residue orientation have been discussed in original research. (D) The metal-dependent mechanism, exemplified by CCoAOMT. Ca²⁺ ions in the active site alter the pK_a of the hydroxyl group and drive nucleophilic attack on the methyl group of SAM. In the stick view, substrates are colored grayish-white; SAM or SAH is colored pink; key residues and metal ions are colored yellow; the distance between S atoms and reactive atoms is labeled as a yellow dashed line; the hydrogen bond is labeled as a red dashed line; and reactive atoms in substrates are indicated by black arrows.

Figure 4

(A) Structural alignment and close-up views of DnrK (PDB: 1TW3) and RdmB (PDB: 1XDS). Variable regions around active sites are colored orange and blue for DnrK and RdmB respectively. Asterisks indicate the “gate” residues (Phe300 in RdmB). (B) Sequence alignment of residues around variable region (R1). The pairwise alignment is performed by EMBOSS needle and edited by ESPript. (C) Close-up views of enzymes in cartoon (top) and surface (bottom) depictions show key residues in the active sites of DnrK, DnrK-S (PDB: 4WXH) and RdmB. Substrates are colored grayish-white; the cofactor SAM or SAH is colored pink; and residues are colored yellow.

Figure 5

Overall dimeric structures (left) and close-up stereo views (right) of (A) TleD (PDB: 5GM2), (B) SpnF (PDB: 4PNE) and (C) LepI (PDB: 6IX5). One subunit in the dimer is colored light yellow and orange; the other is colored light cyan and blue. Additional N-termini acting as lids to compact the active site are underscored in orange and blue in each subunit; the substrate and cofactor are colored grayish-white and pink, respectively; the green circles show the substrate cavity of each enzyme.

Scheme 1

Mechanism of radical-SAM-based reactions. The C–S bond of the SAM molecule is disrupted by homolytic cleavage, and 5ʹ-dAdo∙ is obtained. 5ʹ-dAdo∙ then directly initiates the subsequent reaction or abstracts hydrogen from the second SAM to yield radical SAM and drives the subsequent reactions indirectly.

Scheme 2

Reactions catalyzed by HemN and RdmB. (A) Proposed radical-mediated decarboxylation catalyzed by HemN. (B) The proposed arginine-mediated decarboxylation is catalyzed by both RdmB and DnrK. Hydroxylation and methylation are catalyzed by RdmB and DnrK, respectively. Two possible pathways of oxygenation are shown in the dashed rectangle, and the corresponding atom in RdmB-mediated oxygenation is colored orange; the corresponding atom in DnrK-mediated neutralization and methylation is colored blue. Ovals in gray represent residues in enzymes. Proposed electron transfers are indicated by curved arrows.

Scheme 3

Ring-opening reaction catalyzed by ChuW.

Scheme 4

Cyclization catalyzed by SAM-dependent enzymes. (A) Methylation and cyclopropanation catalyzed by YtkT. (B) Proposed mechanism of radical-mediated cyclopropanation catalyzed by C10P and C10Q. (C) Proposed mechanism of bicarbonate-mediated cyclopropanation catalyzed by CMASs and CFASs. (D) Cyclization and methylation catalyzed by VioH and VioG.

Scheme 5

Multiple reactions catalyzed by the indicated enzymes. (A) Proposed mechanism for the formation of the spiro-ring catalyzed by SlnM. (B) Proposed mechanism of carbocation-mediated cyclization catalyzed by TleD. (C) [4+2] and [6+4] cycloaddition and Cope rearrangement catalyzed by SpnF. (D) Dehydration, branch pericyclization and rearrangement catalyzed by LepI. SAM and its analogs are shown in the box. Ovals in gray represent residues or corresponding enzymes. Proposed electron transfers are indicated by curved arrows. MTA, 5ʹ-deoxy-5ʹ-(methylthio) adenosine; DA, Diels‒Alder reaction; IMDA, intramolecular Diels‒Alder reaction; HAD, hetero-Diels‒Alder reaction.

Scheme 6

Reactions catalyzed by CysG and PsoF. (A) Biosynthesis of siroheme catalyzed by CysG and branch pathways from the intermediates catalyzed by other enzymes. (B) Three reactions catalyzed by PsoF in the biosynthesis of pseurotin. Methylation is separated from isomerization and epoxidation in the biosynthesis cascade. Proposed electron transfers are indicated by curved arrows. PsoF-MT, methyltransferase domain of PsoF; PsoF-FMO, FAD-containing monooxygenase domain.

Figure 6

(A) Homology with CysG and reactions catalyzed by each enzyme. (B) Sequence alignment for CysG^A and CysG^B and their homologies. Some of the shared folds and SAM binding motifs are shown. The alignment is performed by CLUSTALW and edited by ESPript. (C) Overall structure of CysG (PDB: 1PJS). The two domains containing NAD⁺ and SAM in each subunit are shown as domain A and domain B, roughly divided by the dashed line. The light-yellow subunit is labeled with domains I^B, II^B and III^B in domain B, and the light-cyan subunit is labeled with domains I^A and II^A. NAD⁺ is colored purple; SAM is colored pink; phosphor-Ser128 is colored yellow and red.

Figure 7

Structural-based sequence alignment of non-methylating SAM-dependent enzymes shows class I MTases core domains, which catalyzes non-methylations or multiple reactions without radical SAM. Triangles indicate the conserved/partial conserved residues in motifs for SAM binding. The alignment is performed by T-Coffee and edited by ESPript.