architectures, mechanisms and molecular evolution of natural product methyltransferases
TRANSCRIPT
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Cite this: DOI: 10.1039/c2np20029e
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Architectures, mechanisms and molec
ular evolution of natural productmethyltransferases†‡David K. Liscombe,x Gordon V. Louie and Joseph P. Noel*
Received 5th March 2012
DOI: 10.1039/c2np20029e
Covering: up to January 2012
The addition of a methyl moiety to a small chemical is a common transformation in the biosynthesis of
natural products across all three domains of life. These methylation reactions are most often catalysed
by S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTs). MTs are categorized based
on the electron-rich, methyl accepting atom, usually O, N, C, or S. SAM-dependent natural product
MTs (NPMTs) are responsible for the modification of a wide array of structurally distinct substrates,
including signalling and host defense compounds, pigments, prosthetic groups, cofactors, cell
membrane and cell wall components, and xenobiotics. Most notably, methylation modulates the
bioavailability, bioactivity, and reactivity of acceptor molecules, and thus exerts a central role on the
functional output of many metabolic pathways. Our current understanding of the structural
enzymology of NPMTs groups these phylogenetically diverse enzymes into two MT-superfamily fold
classes (class I and class III). Structural biology has also shed light on the catalytic mechanisms and
molecular bases for substrate specificity for over fifty NPMTs. These biophysical-based approaches
have contributed to our understanding of NPMT evolution, demonstrating how a widespread protein
fold evolved to accommodate chemically diverse methyl acceptors and to catalyse disparate
mechanisms suited to the physiochemical properties of the target substrates. This evolutionary diversity
suggests that NPMTs may serve as starting points for generating new biocatalysts.
1 Introduction
2 Methyl acceptor diversity
3 Primary structure of NPMTs and identification of
NPMT genes
4 Structural biology of NPMTs
5 Architecture of SAM binding
6 Structural basis of NPMT substrate specificity
7 Mechanisms of SAM-dependent methylation
8 Evolution of NPMTs
9 Engineering of NPMTs
10 Conclusions and future perspectives
11 Acknowledgements
12 References
Howard Hughes Medical Institute, Jack H. Skirball Center for ChemicalBiology and Proteomics, Salk Institute for Biological Studies, La Jolla,CA 92037, USA. E-mail: [email protected]
† This paper is part of an NPR themed issue on Structural Aspects ofBiosynthesis.
‡ In memory of Prof. Dr Joachim Schr€oder and his contributions to ourunderstanding of plant metabolism.
x Current address: Vineland Research and Innovation Centre, VinelandStation, ON, Canada; e-mail: [email protected]
This journal is ª The Royal Society of Chemistry 2012
1 Introduction
Methylation is a ubiquitous biotransformation in nature, used
throughout all branches of metabolism and often key to meta-
bolic homeostasis. These transformations modulate diverse
biological processes such as cell signaling, and the biosynthesis of
complex and sometimes unique specialized metabolites. These
reactions are most often catalysed by methyltransferases [MTs;
sometimes referred to as (trans)methylases] that rely on the co-
substrate{ S-adenosyl-L-methionine (SAM 1; Fig. 1) as an elec-
tron-deficient methyl donor. The by-product of methylation is
S-adenosyl-L-homocysteine (SAH 2; Fig. 1). Small molecule, or
natural product, methyltransferases (NPMTs) participate in the
biosynthesis and modification of bioactive molecules derived
from several branches of primary and secondary (specialized)
metabolism, including membrane components,1,2 cofactors,3
prosthetic groups,4 pigments,5,6 and signaling7,8 and defense
compounds.9–11
{ While often classified as a cofactor, herein we use co-substrate todescribe SAM, because SAH is catabolized and SAM is notregenerated after methyl transfer.
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Since the first X-ray crystal structure of a NPMT12 reported
early in the 1990’s, dozens of structures have been elucidated for
NPMTs representing a chemically diverse array of methyl
acceptors. Surprisingly, all currently known NPMT structures
belong to either of two (Class I and Class III)13 of 15 currently
recognized protein fold superfamilies of SAM-binding
proteins.14 Even so, the large majority belong to the Class I or
Rossmann-like fold family.15 The structural elucidation of
NPMTs advanced our understanding of both the molecular
determinants for substrate specificity and the varied catalytic
mechanisms of this class of enzymes, while also providing a
foundation for structure-based engineering to generate new
enzymes with altered specificities. Macromolecular (DNA,
Gordon V: Louie
Gordon Louie obtained B.Sc.
Honours (Biochemistry and
Chemistry) and Ph.D.
(Biochemistry) degrees from
the University of British
Columbia. He undertook post-
doctoral research in the labora-
tories of Prof. Thomas Blundell
at Birkbeck College, Univ. of
London (where he determined
the structure of a porphobili-
nogen deaminase, a key enzyme
in tetrapyrrole biosynthesis),
and Prof. Senyon Choe at the
Salk Institute for Biological
Studies (where he characterized
the interaction of diphtheria toxin with its receptor). He subse-
quently worked as a structural biologist at SGX Pharmaceuticals.
Gordon is presently a Research Associate in Prof. Joseph Noel’s
lab at the Salk Institute, focusing on structural analyses of enzymes
of phenylpropanoid metabolism.
David K: Liscombe
David Liscombe completed the
Honours Biology and Pharma-
cology Co-op program at
McMaster University in 2003.
He received his Ph.D. in plant
biochemistry from the Univer-
sity of Calgary in 2008, which
involved the characterization of
methyltransferases from benzy-
lisoquinoline alkaloid biosyn-
thesis. As a postdoc in Sarah
O’Connor’s lab at MIT, David
discovered genes involved in
terpenoid indole alkaloid
biosynthesis. He subsequently
moved to Joseph Noel’s lab at
the Salk Institute to investigate the structural biology of methyl-
transferases and other specialized biosynthetic enzymes. David is
currently a Research Associate in Applied Genomics at Vineland
Research and Innovation Centre in Ontario, Canada.
Nat. Prod. Rep.
RNA, and protein) MTs are essential epigenetic regulators of
gene expression and chromatin structure, and post-translational
modulators of protein function. The structures and functions of
macromolecular MTs are reviewed elsewhere.13,15 In this review,
we discuss the structure, function, and evolution of SAM-
dependent NPMTs, focusing on those with structures reported
in the literature. A compilation of NPMTs with published
structures is summarized in Table 1.
2 Methyl acceptor diversity
All MTs (EC 2.1.1.-) are classified according to the substrate
atom that accepts the methyl group, usually O (54% of EC
subclass), N (23%), or C (18%). S-directed MTs (3% of EC
subclass) and NPMTs that accommodate other acceptors (such
as halides; 2%) are rare but notable,16–18 and some NPMTs
transfer a methyl moiety to more than one type of acceptor
atom.19
Considering all domains of life, the most abundant NPMTs
are O-directed MTs (OMTs). The OMT subfamily in certain
plants and bacteria has undergone tremendous genetic and
functional expansion. For example, poplar trees (Populus sp.)
encode 26 small molecule OMTs,20 whereas only two OMTs are
found in humans [catechol OMT (COMT) and N-acetyl-sero-
tonin OMT] and yeast [Saccharomyces cerevisiae; trans-aconitate
methyltransferase (TMT1)21 and cantharidin resistance gene
(Crg1)22]. Humans and yeast are not remarkable sources of
complex natural products, however, so their lack of encoded
OMT genes is not surprising as small molecule OMTs participate
almost exclusively in specialized metabolic pathways. Hydroxyl
moieties of phenolics, such as catechol, and hexoses, along with
carboxylic acids or CoA esters are the most common substrates
for NPMTs (Fig. 2). OMTs tend to be regiospecific, but some,
such as those involved in phenylpropanoid and flavonoid
biosynthesis in photosynthetic organisms, are less selective,
Joseph P: Noel
Joseph Noel obtained a Bach-
elor of Science degree in Chem-
istry from the University of
Pittsburgh at Johnstown in
1985. He received his Ph.D. in
chemistry from the Ohio State
University in 1990, working on
the enzymology of phospholi-
pases with Professor Ming-Daw
Tsai. As a postdoctoral fellow
with the late Paul B. Sigler in
the Department of Molecular
Biophysics and Biochemistry at
Yale, Joe elucidated the struc-
ture of heterotrimeric G-
proteins. Joe is currently
director of the Jack H. Skirball Center for Chemical Biology and
Proteomics at the Salk Institute, professor at the Salk Institute and
investigator with the Howard Hughes Medical Institute.
This journal is ª The Royal Society of Chemistry 2012
Fig. 1 S-Adenosyl-L-methionine (SAM) the methyl donor for SAM-
dependent natural product methyltransferases (NPMTs). (A) NPMTs
use SAM co-substrate as a reactive electron deficient methyl group
(green) donor for transfer to a electron-rich methyl acceptor (Nu:). (B) In
addition to the methylated product, S-adenosyl-L-homocysteine (SAH)
forms and is a potent inhibitor of SAM-dependent MTs. (C) Sinefungin,
a fungal-derived SAM-analog possessing an amine group (red) in place
of the methylsulfonium moiety serves as a competitive inhibitor of
SAM-dependent MTs.
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capable of sequential methylations of the same or similar
substrates.23–25
Natural product N-directed MTs (NMTs), though not as
numerous as OMTs, are broadly represented across all domains
of life. NMTs are commonplace in signal transduction pathways
in animals, where they modulate the activity of signaling mole-
cules [COMT, phenylethanolamine NMT (PNMT), histamine
NMT (HNMT), indolethylamine NMT (INMT)]. There are also
a few examples of iterative NMTs. Plasmodium falciparum
phosphoethanolamine NMT (PfPEANMT) catalyzes the
This journal is ª The Royal Society of Chemistry 2012
trimethylation of phosphoethanolamine in phosphocholine
biosynthesis.2 Caffeine biosynthesis in certain plants employs a
bifunctional NMT, dimethylxanthine NMT (DXNMT), which
performs sequential methylations at two N-containing sites.26
Similarly, bacterial NMTs TylM1 and DesVI catalyze analogous
dimethylations of hexosamine moities.27,28 Characterized natural
product NMTs are collectively responsible for the methylation of
a wide variety of substrates, such as non-ribosomal peptides
(both peptide bonds and side chains), hexosamines, primary
amines, secondary amines (i.e. indoles, imidazoles, more complex
alkaloids), and tertiary amines (Table 1, Fig. 3).
Small molecule C-methyltransferases are most often found in
bacterial and plant systems, and are relatively scarce in other
branches of the eukaryotic lineage. Yeast encodes only two,21
and no small molecule CMTs have been detected or predicted to
occur in humans. Like OMTs, the known CMTs participate
primarily in specialized metabolism (Fig. 3), methylating
substrates such as tetrapyrroles, phenolics, aliphatics, and
hexos(amin)es. We also consider cyclopropane synthases as
SAM-dependent MTs, although they technically transfer a
methylene group.1
The few S-directed MTs identified and characterized to date
exist in plants and mammals. In plants, they produce
volatile halogen and sulfur compounds or biosynthetically tailor
thiocyanates.16,29,19,30,31 Human thiopurine S-MTs (TPMT)
participate in the detoxification of xenobiotics.18
3 Primary structure of NPMTs and identification ofNPMT genes
The NPMTs vary in length, typically spanning 200–500 amino-
acid residues corresponding to monomeric molecular masses of
ca. 25–55 kDa. Almost all NPMTs isolated to date possess an a/b
structure: alternating a-helices and b-strands along the length of
the polypeptide chain. Comparative sequence analyses of SAM-
dependent MTs have identified a series of conserved motifs
shared among these proteins.14,32–35 Generally arranged in
sequential order across the core MT domain, Motifs I–VI reside
in regions associated with SAM co-substrate binding (Fig. 4).
These motifs are widely conserved across NPMTs, albeit to
varying degrees, and are considered to be defining features of
SAM-dependent MTs.14,32,33,36,37 Kozbial and Mushegian (2005)
provide the most recent survey of conserved motifs in MTs.14
Motif I is present in the majority of MTs (69 of 84 MT proteins
surveyed32) and is often used for the initial bioinformatic iden-
tification of putative MTs. This motif spans the loop preceding
the first b-strand (b1) of the core Rossmann fold leading into
the following a-helix (aA). It includes a nine-residue amino
acid block with the consensus sequence (V/I/L)(L/V)(D/E)(V/I)-
G(G/C)G(T/P)G.32 This nine-residue structure incorporates the
glycine-rich ‘‘GxGxG’’ signature sequence, a SAM-binding motif
found in almost all SAM-dependent MTs.14,32,35,38 Although
none of the three glycines of the GxGxG motif is universally
conserved, substitutions typically encompass small
sidechains.14,38
Motif II spans b2 and the adjoining turn. Here, the consensus
sequence in plant OMTs (aka Motif B35) differs from that
observed in a larger survey of functionally-diverse MTs.32 Only
two residues, DA, are common to both consensus sequences,35
Nat. Prod. Rep.
Table 1 Published tertiary structures of natural product methyltransferases
Name Organism Acceptora PathwaySubstrateclass Foldb
Structuralsimilarityc Mechanismd
Metal-dependent? PDBe Ref.
BACTERIALDnrK Streptomyces peucetius O, 5 daunorubicin phenolic I, D RdmB PD No 1TW2 80RebM Lechevalieria
aerocolonigenesO, 6 rebeccamycin hexose I, M CPFASs,
DnrKAB No 3BUS 86
SynOMT Synechocystis sp. strainPCC 6803
O, 12 hydroxycinnamicacids
phenolic I, D PFOMT,COMT,
M Yes 3CBG 24
BcOMT2 Bacillus cereus O, 14 ? phenolic I, D CCoAOMT,COMT
M Yes 3DUL 79
NcsB1 Streptomycescarzinostaticus
O, 11 neocarzinostatin phenolic I, D DnrK, RdmB, AB No 3I53 85
DhpI Streptomyces luridus O, 19 dehydrophos phosphoryl I, D NNMT ? No 3OU2 87NovP Streptomyces spheroides O, 21 novobiocin hexose I, D COMT,
SynOMTAB(M) Yes 2WK1 78
CalO1 Micromonosporaechinospora LL6600
O, 16 calicheamicin phenolic I, D ChOMT,MmcR
AB No 3LST 88
MmcR Streptomyces lavendulae O, 18 mitomycin quinone/mitosane
I, D DnrK, NcsB1 AB No 3GWZ 89
MycE Micromonosporagriseorubida
O, 20 mycinamicin hexose I, T COMT AB(M) Yes 3SSM 50
PhzM Pseudomonas aeruginosa N, 22 procyanin phenazine I, D CaOMT,IOMT
PD? No 2IP2 90
DesVI Streptomyces venezuelae N, 23 erythromycin hexosamine(primary)
I, D GNMT,TylM1
PD No 3BXO 28
MtfA Amycolatopsis orientalis N, 25 chloroeremomycin primaryamine
I, D PhzM, DesVI AB No 3G2M 84
NodS Bradyrhizobium japonicum N, 28 nodulation factor hexosamine(primary)
I, M DhpI AB No 3OFJ 91
TylM1 Streptomyces fradiae N, 24 tylosin hexosamine(primary)
I, D DesVI,GNMT
AB? No 3PFG 27
CbiF Bacillus megaterium C, 43 vitamin B12 tetrapyrrole III,D CobA PD No 1CBF 3CPFASs Mycobacterium
tuberculosisC, 38 cyclopropyl lipids aliphatic I, D RebM AB No 1KP9 1
CbiT Methanothermobacterthermautotrophicus
C, 41 vitamin B12 n/a I, T COMT n/a n/a 1KXZ 54
CysG Salmonella enterica C, 41 siroheme tetrapyrrole III,D
CobA AB No 1PJQ 4
CobA Pseudomonas denitrificans C, 41 tetrapyrroles tetrapyrrole III,D
CbiF AB No 1S4D 56
BchU Chlorobium tepidum C, 41 bacteriochlorophyllc
tetrapyrrole I, D PhzM,LpCaOMT
AB No 1X19 53
MtCbiL Methanothermobacterthermoautotrophicus
C, 41 tetrapyrroles tetrapyrrole III,D
CbiF, CobA AB No 2QBU 57
CbiL Chlorobium tepidum C, 41 vitamin B12 tetrapyrrole III,D
CbiF, CobA AB No 2E0K 55
TcaB9 Micromonospora chalcea C, 39 D-tetronitrose hexosamine I, M 3DLI AB No 3NDI 52GPPMT Streptomyces lasaliensis C, 40 2-methylisoborneol aliphatic (I) RebM ? ? n/a 92NirE Pseudomonas aeruginosa C, 41 heme d1 cofactor tetrapyrrole III,
DCobA AB No 2YBO 58
LiOMT Leptospira interrogans ? ? ? I, D CCoAOMT M (Yes) 2HNK 93RdmB Streptomyces purpurascens n/a anthracycline tertiary
carbonI, D IOMT, DnrK n/a No 1QZZ 37,
75
PLANTChOMT Medicago truncatula O, 10 chalcone phenolic I, D CaOMT,
IOMTAB No 1FPQ 5
I7OMT Medicago truncatula O, 8 isoflavone phenolic I, D CaOMT,ChOMT
AB No 1FPX 5
MtCaOMT Medicago sativa O, 13 phenylpropanoid phenolic I, D I7OMT,CalO1
AB No 1KYW 77
SAMT Clarkia breweri O, 7 salicylic acidsignalling
phenolic I, D DXMT,XMT, HNMT
PD No 1M6E 7
CCoAOMT Medicago sativa O, 18 hydroxycinnamyl-CoAs
phenolic I, D COMT M Yes 1SUI 51
HIOMT Medicago truncatula O, 8 isoflavone phenolic I, D I7OMT? AB No 1ZHF 9PFOMT Mesembryanthemum
crystallinumO, 15 phenylpropanoid phenolic I, D CCoAOMT M Yes 3C3Y 25
IAMT Arabidopsis thaliana O, 9 indole acetic acid carboxy(indole)
I, D SAMT PD No 3B5I 74
LpCaOMT Lolium perenne O, 13 phenylpropanoid phenolic I, D AB No 3P9C 23
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Table 1 (Contd. )
Name Organism Acceptora PathwaySubstrateclass Foldb
Structuralsimilarityc Mechanismd
Metal-dependent? PDBe Ref.
I7OMT,CalO1
AtHOL1 Arabidopsis thaliana NCS- > I >Br > Cl
thiocynates, halides(not F)
thiocyanate/halide
I, D TPMT ? No 3LCC 19
DXMT Coffea canephora N, 27, 29 caffeinebiosynthesis
purine I, D SAMT PD No 2EFJ 26
XMT Coffea canephora N, 26 caffeinebiosynthesis
purine I, D SAMT PD No 2EG5 26
ANIMALTPMT Mus musculus S, 42 thiopurine
xenobioticsthiopurine I, M PNMT,
HNMTAB No 3BGD 18
COMT Rattus norvegicus O, 4 catechol phenolic I, M PFOMT AB/M?14 Yes 1VID 12GNMT R. norvegicus N, 30 glycine primary
amineI, T TylM1 AB No 1XVA 72
HNMT Homo sapiens N, 31 histamine imidazole I, M TPMT PD No 1JQD 94PNMT H. sapiens N, 32 adrenaline primary
amineI, M GNMT PD No 1HNN 8
GANMT R. norvegicus N, 34 creatine secondaryamine
I, D COMT,GNMT
AB No 1KHH 95,96
PfPENMT Plasmodium falciparum N, 35 phosphocholine primary/s/tertamine
I, D CPFASs AB No 3UJ6 2
NNMT H. sapiens N, 33 nicotinamide pyridine I, M PNMT,INMT
PD No 3ROD 73
a Acceptor atom, compound number in Fig. 2–3. b Class, oligomerization state (M, monomer; D, dimer; T, tetramer). c Structural homologs in italicswere determined using the DALI server (http://ekhidna.biocenter.helsinki.fi/dali_server/), or from PDB records at http://www.rcsb.org. d PD, proximityand desolvation effects; AB, acid–base; AB(M) acid–base with possible metal participation; M, metal-dependent. e Representative PDB code provided.
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with the aspartate residue reflecting the conservation of an acidic
residue near the C-terminus of b2.
Motif III spans b3, followed by Motif IV spanning b4 and the
adjoining loops. Both motifs include a partially-conserved acidic
residue at the C-terminus.14 Motif V, occurring in the helix (aC)
following Motif IV, sometimes harbors hydrophobic residues
that sandwich the adenine moiety of SAM.14 A well-conserved
glycine residue is characteristic of Motif VI, which corresponds
to b5 and the loop between aC and b5.14 Some NPMTs contain
multiple MT domains,39 or contain heterologous domains that
catalyze reactions other than methylation.4
Many NPMT genes have been isolated based on these
conserved motifs. PCR-based screening with degenerate primers
restrained to the Motif I consensus sequence have been used to
amplify fragments of NPMT genes.40–42 Transcriptome and
genome sequencing projects often rely on these sequence signa-
tures and homology to known MTs as a means of isolating
previously undiscovered NPMT genes.10,42,43 Computational
methods based on motif scanning and/or structural homology
have been developed for the identification and characterization
of MT-encoding genes, to elucidate the ‘methyltransferome’ in
whole genomes20,21,44–47 and to define MT domains within
biosynthetic gene clusters.38
4 Structural biology of NPMTs
Protein X-ray crystallography has been used extensively over the
last two decades to determine the tertiary and quaternary
structures of numerous NPMTs (Table 1). The first MT structure
solved was the cytosine C5-specific DNA MT, M.HhaI.48 The
This journal is ª The Royal Society of Chemistry 2012
SAM binding domain of M.HhaI incorporates the Rossmann
fold,48 an a/b domain well known for binding nucleotide-con-
taining cofactors, such as NAD.49 Soon after, the first structure
of an NPMT was reported,12 and surprisingly, the fold of cate-
chol OMT (COMT; Table I1) was strikingly similar to the DNA
MT. Indeed, both enzymes include a core seven b-strand Ross-
mann fold, which serves as a SAM-binding domain. Further-
more, structural comparison of M.HhaI, COMT, and NAD-
dependent alcohol dehydrogenases demonstrates that the
respective Rossmann folds and bound nucleotide-based cofactor/
co-substrate superimpose remarkably well (Fig. 5).
The similarity between SAM binding domains of M.HhaI and
COMT suggested initially that all MTs might share a common
structure.12 The so-called Class I, Rossmann-like MT fold is
shaped by the alternating a-helices and b-strands of the poly-
peptide (Fig. 4, 6a), which form a relatively planar b-sheet
sandwiched by a-helices (Fig. 6b). The N-terminal b-strand
inserts into the middle of the b-sheet, such that the strand
topology is 3214576, with the seventh strand antiparallel to all
other strands (Fig. 5a). The functionally important, conserved
residues of the substrate-binding and catalytic sites are typically
located in the C-terminal regions of b-strands or in the adjoining
loops. This core fold is often elaborated by additional helices or
b-hairpins.
Since the structure of COMT was reported, more than 50
NPMT structures have been solved and published (Table 1), and
even more structures have been deposited in the Protein Data
Bank (PDB) but not yet described in the literature. These anal-
yses further establish the structural conservation of the core of
most NPMTs, and additionally delineate the structural diversity
Nat. Prod. Rep.
Fig. 2 Chemical diversity of natural product OMT substrates. Compounds are numbered, and named as appropriate. Methylation target sites are
highlighted in green. Structurally-characterized NPMTs and their substrate numbers are indicated in Table 1.
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of the embellishing domains appended to or inserted into the
primary structure of the core Rossman-like fold. Most
commonly, the additional domains represent N-terminal exten-
sions that mediate oligomerization (quaternary structure) and/or
modulate substrate specificity.14
For a number of NPMTs, catalytic activity is dependent on a
coordinated divalent cation (Table 1), such as Mg2+ (magne-
sium)51 or Ca2+ (calcium),52 and structural studies have eluci-
dated the architecture and functional role of the metal binding
site. Mg2+- and Ca2+-dependent NPMTs typically use an acidic
triad [DD(D/N)] for metal coordination. A zinc-containing
NPMT uses four cysteine residues,53 although metal binding
occurs distal to the active site and may serve only in structure
stabilization. As best exemplified by the metal-dependent OMTs,
divalent cations typically participate directly in substrate binding
and/or catalysis.
The initial presumption that all NPMTs share the same
structural core, a Rossmann-like fold, was disproved with the
structural characterization of cobalt-precorrin-4-MT (CbiF) and
the discovery of the Class III MT fold.3 CbiF and five other
closely related MTs act sequentially in the biosynthesis of the
corrin ring of vitamin B12 (a tetrapyrrole) in bacteria. To date,
the Class III fold associates with tetrapyrrole MTs only.
Conversely, other NPMTs known to methylate tetrapyrrole-
Nat. Prod. Rep.
containing substrates, namely BchU53 and CbiT,54 belong to the
Class I fold.
CbiF and its Class III relatives are more closely related to the
GTPase fold typified by a kidney-shaped arrangement of two a/b
domains linked by a single coil (Fig. 6).3,4,55–58 While there is no
topological similarity between the two a/b domains, both contain
a five-stranded b-sheet sandwiched by four a-helices (Fig. 6). A
‘‘GxGxG’’ motif is located in the C-terminal end of b1 and the
loop leading to aA of Class III NPMTs, a region that is also
ascribed to SAM binding3 in this family of tertiary structures.
5 Architecture of SAM binding
In all Class I NPMTs structurally characterized to date, SAM
occupies the spatially equivalent position along the C-terminal
end of the core b-sheet of the Rossmann-like domain (Fig. 7A,B),
despite only weak conservation of the SAM-binding residues.
The Class I NPMTs bind SAM (or SAH) in an extended
conformation maintained by a network of hydrogen bonds and
van der Waals interactions5 involving residues alongMotifs I–III
(Fig. 7A,B). One or more residues in the GxGxG motif are in
contact with the carboxypropyl portion of SAM, while the
conserved acidic residue in Motif II forms hydrogen bonds with
the ribosyl moiety (Fig. 7B). Variable residues in the C-terminal
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Fig. 3 Chemical diversity of natural product N-, C-, and S-directed MT substrates. Compounds are numbered and named. Methylation sites are
highlighted in green. Structurally-characterized NPMTs and their substrate numbers are indicated in Table 1. Due to the size and complexity of
tetrapyrrole substrates, the regiospecificities of uroporphyrinogen-like tetrapyrrole MTs (CysG, NirE, CobA, BchU, and CbiL) are indicated using a
tetrapyrrole scaffold for illustrative purposes (compound 41).
Fig. 4 A schematic diagram of the primary and secondary structure of a
typical NPMT, emphasizing conserved motifs used to identify a putative
MT. N- and C- termini are shown in black circles. a-Helices are shown in
red, b-strands in yellow, and adjoining loops are green. Conserved resi-
dues are stacked for each motif (I–VI), and the highly-conserved
‘‘GxGxG’’ motif is in bold fonts.
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region of b2 (motif II) and the conserved acidic residue in b3
interact with the adenosyl moiety, while variable residues
C-terminal to b4 (Motif IV) appear to contact the amino and
sulfonium groups in the methionine fragment of SAM.14,32
Structure comparison of a number of metal-independent,
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homodimeric plant OMTs reveals substantial flexibility in the
connection between the SAM binding domain and the central
core of the homodimer, which is formed by oligomerization and
substrate-binding domains.23 Thus, the MTs of this lineage
apparently utilize an open conformation for facilitating entry of
SAM and the phenolic substrate (and exit of the reaction prod-
ucts), and a closed conformation for establishing the catalytically
appropriate positioning of substrate and co-substrate prompting
transmethylation.23,85
All Class III MTs bind SAM in a jack-knifed conformation
such that, as Schubert and colleagues describe, SAM/SAH fits
into its binding site ‘‘similar to a two-pronged plug in a socket’’
(Fig. 7C,D).3 It is thought that this conformation might promote
presentation of the methyl group to the bulky precorrin
substrate.3 The SAM-binding site of Class III MTs involves side-
chain and main-chain interactions with residues located on the
b1-aA segment, the polypeptide loop linking b4 and aD, and aE
residues. These three sections line the large trough separating the
N- and C-terminal domains.3
Nat. Prod. Rep.
Fig. 5 The core fold of Class I NPMT incorporates a Rossmann fold.49
Similarly oriented structures of catechol OMT (A) with its bound
cosubstrate SAM (grey spheres), and cofactor-binding domain of alcohol
dehydrogenase (B; PDB: 6ADH, chain A), with bound NAD (grey
spheres), displayed side-by-side. Helices are shown in red, beta-strands in
yellow, and loops in green. Structures were initially superimposed using
the SSM superpose function in Coot.97 Image was generated with
MacPyMol.
Fig. 6 Topologies and folds of NPMTs. (A) Typical topology of a Class
I, Rossmann-like NPMT. (B) Tertiary structure of COMT with bound
SAH, representative of the Class I fold. (C) Topology and (D) tertiary
structure of CbiF with bound SAH illustrate the typical Class III
NPMTs. The diverse N-terminal region of Class I NPMTs is shown in
grey. The SAM/SAH binding site is highlighted in orange, bound SAH
ligands are shown in light blue, and conserved motifs are indicated with
Roman numerals (I, II, III). a-Helices are shown in red, b-strands in
yellow, and adjoining loops are green.
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The by-product of methylation, SAH 2 (Fig. 1), is a potent
inhibitor of all SAM-dependent methyltransferases. In fact,
SAM binding sites in MT crystal structures are often occupied by
SAH, either intentionally through co-crystallization or crystal
soaks with SAH, or as a result of SAM degradation or enzyme-
catalyzed transmethylation. The binding affinity of SAH has
been exploited for MT isolation.59–61 Sinefungin 3, a fungal-
derived SAM analog and a competitive inhibitor of SAM-
dependent MTs,62–66 is also regularly employed in biochemical
and structural investigations of NPMTs. In vitro assays of MTs
can be adversely affected by the accumulation of the inhibitory
SAH by-product. Recently developed coupled assays of MT
activity utilize SAH-catabolizing enzymes, which act both to
prevent SAH accumulation and to generate a free thiol (homo-
cysteine) that can be quantified spectrophotometrically.67,68
Because the interaction of SAM with MTs typically does not
involve the donor methyl group, MTs can also readily accept
chemically generated S-substituted SAH analogs. Thus, as first
demonstrated with DNA MTs,69 NPMTs can utilize SAM
analogs and catalyze the transfer of a non-natural functional
group to a suitable acceptor atom.70,71
6 Structural basis of NPMT substrate specificity
NPMTs are capable of methylating an expansive repertoire of
substrates representing a diversity of chemical scaffolds found in
nature (Fig. 2–3). In contrast to DNA MTs, which possess some
conserved motifs for recognition of common features of the
macromolecular substrates,15 small molecule MTs do not appear
to possess widely conserved structural determinants for methyl
acceptor recognition. Instead, the core Rossmann-like fold of
Class I methyltransferases often bear structural elaborations,
including N-terminal extensions, discrete domains and active-site
caps. These structural appendages are critical determinants of the
functional evolution of MTs as they typically include amino acid
residues that contribute substantially to substrate binding and
the positioning of the methyl accepting atom.7,72,73 For example,
with most metal-independent, homodimeric plant OMTs, the
Nat. Prod. Rep.
N-terminal domains of the polypeptide chain are responsible
for substrate binding and dimerization.5,23,78 In these OMTs, the
C-terminal SAM-binding domain nevertheless plays a key role in
fully sequestering the substrate upon formation of the catalyti-
cally primed and closed conformational state.5,23,78 Indeed, non-
productive ligand complexes observed with alfalfa CaOMT77
may be representative of exploratory ‘‘pre-binding’’ modes of the
incoming substrate molecule with CaOMT that is in an open
conformational state and lacking a fully formed phenolic-
substrate binding pocket.23 Such considerations highlight the
importance of conformational dynamics for the substrate-
binding and catalytic activities of the MTs. Interestingly, heter-
odimerization among four distinct but closely related OMTs
involved in berberine biosynthesis can yield a substrate-speci-
ficity profile different from that of any of the four homodimeric
enzymes.41
Structural biology has contributed to our understanding of
substrate discrimination within NPMT subfamilies that are
known to accept similar chemical scaffolds, structure–function
relationships that can be understood only in the context of
appropriately configured active site architectures.7,9,23,74 Detailed
structural analyses of NPMTs bound to substrates, products, or
analogs have shed light on the enzymatic determinants of
substrate preferences.5,9,23For example, CaOMT possesses broad
This journal is ª The Royal Society of Chemistry 2012
Fig. 7 Binding modes of SAM NPMT active sites. (A) Superimposed
structures of a representative set of Class I NPMTs, CCoAOMT (grey)
with bound SAH (yellow), and SAMT (green) with bound SAH
(magenta) illustrating the canonical SAM/SAH binding site in Class I,
Rossmann-like NPMTs. (B) Zoomed-in view of SAM/SAH binding sites
using the same color scheme as (A). Only b-strands (b1-4) and adjoining
loops involved in co-substrate binding are depicted. Conserved motifs are
labeled, and the conserved acidic residues (in this case, both are Asp
residues) in Motif II are shown as sticks and labeled with an asterisk. (C)
Superimposed structures of CbiF (orange) with bound SAH (yellow), and
CobA (blue) with bound SAH (cyan) illustrating SAM/SAH binding sites
typical of Class III NPMTs. (D) Close-up view of Class III SAM/SAH
binding sites using same color scheme as (C). Residues within 4 �A of SAH
are shown, illustrating the conservation of the co-substrate binding site in
the Class III MT folds. Structures were superimposed using the SSM
superpose function in Coot.97 Images were generated with MacPyMol.
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substrate selection (in vitro), with bi-functional activity in meta-
O-methylation of both the 3- and 5-hydroxyl groups of phenolic
monolignol-precursors bearing alcohol, aldehyde, or acid forms
of the propenyl side group. The interactions dictated by the
substrate’s C3 and C9(g) substituents likely govern the substrate-
preference patterns of the CaOMTs.23 In particular, for angio-
sperm CaOMTs, occupancy of a predominantly hydrophobic
cavity by a C3 methoxy-substituent on the substrate imposes a
preference for 5-O-methylation activity, while the presence of
only one hydrogen-bonding residue near the substrate’s C9
functionality underlies the preference for the singly oxygenated
alcohol and aldehyde forms of the monolignol precursor.
The initial characterization of putative NPMTs is challenging
due to the unknown identity of the true in vivo substrate(s) and
the unavailability of synthetic substrates suitable for assays and
further functional studies. Homology, or even substantial
sequence similarity, does not necessarily reveal useful informa-
tion as to the nature of the methyl acceptor or substrate
This journal is ª The Royal Society of Chemistry 2012
specificity of an orphan NPMT. For example, an NMT involved
in terpenoid indole alkaloid biosynthesis in Madagascar peri-
winkle (Catharanthus roseus) is unexpectedly most similar to
g-tocopherol CMTs involved in vitamin E biosynthesis. Indeed,
these enzymes methylate structurally and chemically disparate
substrates (36 versus 37 in Fig. 3) originating in very distinct
realms of metabolism.10
Phylogenetic analyses can assist in predicting substrate speci-
ficity, but even close phylogenetic relationships can be
misleading. For example, RdmB is phylogenetically and struc-
turally related to the OMT DnrK, but RdmB functions as a
hydroxylase.37,75 Furthermore, there are no obvious trends in
mechanistic strategies of NPMTs with respect to acceptor spec-
ificity (Table 1), except that NMTs and CMTs are rarely, if ever,
metal-dependent. This presence or absence of metal cation
binding further illustrates how structural enzymology can
provide important clues to facilitate the rapid elucidation of
substrate selection. Thus, even the most readily discernable
structural information, primary structure, can also provide
some hints, based on the presence of metal-coordinating motifs
(i.e. DDD/N).
Finally, the development of a substrate tagging approach
using SAM analogues and crosslinking offers additional exper-
imental methods for elucidating substrate specificity.70 Targeted,
activity-based metabolite profiling has been employed to identify
in vivo substrates for NPMTs,10 and a systems biology approach
has efficiently elucidated the biological functions of an orphan
NPMT.22
7 Mechanisms of SAM-dependent methylation
NPMT-catalyzed transmethylation occurs via SN2-like nucleo-
philic substitution,76 and detailed structural analyses of NPMTs
reveal several requisite features of this reaction. Firstly, the
active-site architecture of the NPMTmust ensure that the methyl
acceptor is positioned reasonably close to the donor electron
deficient methyl group of SAM, usually within about 3 �A (to
methyl carbon, or ca. 4 �A between acceptor and sulfonium
moiety; Fig. 8). In addition, the acceptor must serve as the most
chemically reactive and spatially proximal nucleophile in the
vicinity of the electron deficient methyl moiety, a consideration
that may demand the enzymic activation of the acceptor through
proton abstraction or in some cases the extrusion of water. As
such, NPMTs have evolved three currently recognized chemical
mechanisms for catalyzing the transmethylation reaction: (i)
proximity and desolvation, (ii) general acid/base-mediated
catalysis, and (iii) metal-dependent mechanisms (Fig. 8).
A primary mechanistic role for proximity and desolvation
(PD, Fig. 8A) in an NPMT-catalyzed transmethylation reaction
was first posited with the structural characterization of SAMT, a
defining member of the SABATH (Salicylic Acid, Benzoic Acid,
THeobromine synthase) family of plant MTs.7 The PD mecha-
nism does not require the direct participation of a catalytic
group(s) from the enzyme, but rather the architecture and
chemical environment of the enzyme active-site ensure that the
acceptor is in close proximity to the donor methyl group and
suitably oriented for nucleophilic substitution, and that water
(solvent) molecules are excluded from the donor–acceptor
interface (desolvation).7 Many other MTs also appear to utilize
Nat. Prod. Rep.
Fig. 8 Catalytic strategies for methylation used by NPMTs. (A) The
‘‘proximity and desolvation effects’’ mechanism, exemplified by DnrK-
mediated anthracycline O-methylation.80 The product, 4-methoxy-3-
rhodomycin T (magenta), and SAH (yellow) from a ternary complex with
DnrK are shown. Mutagenesis of the closest possible general base, Y142
(grey), did not have a substantial effect on catalytic rate. The methylated
oxygen, which is in proximity (distance in green) to the sulfonium group
of SAM is indicated with an arrow. (B) The ‘‘acid-base’’ mechanism of
methyl-transfer demonstrated by PfPEANMT.2 His and Tyr residues
(green) work in-concert as a ‘general base’ to deprotonate the substrate,
in this case phosphoethanolamine (magenta) driving the SN2-transfer of
the methyl group from SAM (yellow), which is proximal to the acceptor
nitrogen (distance shown in green). (C) Metal-dependent methylation
catalysis illustrated by CCoAOMT. Metal-mediated deprotonation of
the acceptor hydroxyl group generates an oxyanion adjacent (distance in
green) to the reactive and electron deficient methyl group of SAM,
Nat. Prod. Rep.
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this strategy (i.e.DnrK, Fig. 8A), irrespective of methyl acceptor
or substrate specificity (Table I).
General acid/base-mediated transmethylation (Fig. 8B) by an
NPMT typically involves an essential catalytic residue that acts
as a general base to deprotonate and thereby activate the methyl
acceptor for nucleophilic attack on the reactive methyl group of
SAM.5,77 Adjacent active-site residues often ensure optimal
orientation of the catalytic base, or work in concert to form a
proton shuttle system (Fig. 8B).2,5 The cyclopropane synthases
(CPFASs)1 employ a carbonate ion as a general base, which may
deprotonate the methyl group of the carbocation intermediate
and thus enable formation of the cyclopropane product.
Conserved residues involved in carbonate-ion binding seem to
distinguish methylene transferases from other NPMTs.14
There appear to be two types of metal-dependent NPMTs.
First are those that rely on a divalent cation solely for substrate
coordination and require a nearby residue as a general base for
deprotonation of the methyl acceptor (i.e. hexose MTs like
NovP,78 and MycE50). Metal-dependent NPMTs of the second
type (almost exclusively phenolic OMTs from plants) use a
catalytic mechanism (Fig. 8C) in which the metal ion perturbs the
pKa of the substrate’s phenolic hydroxyl group and thus
promotes loss of the hydroxyl proton (as a hydronium ion) and
formation of a nucleophilic phenolate anion.24,25,51,79 Such a
reaction mechanism was originally proposed for COMT, but
subsequent studies suggest that an active-site lysine residue is
responsible for deprotonation of the acceptor, indicative of a
general acid/base mechanism.15
8 Evolution of NPMTs
Kozbial andMushegian suggested that the last universal common
ancestor of cellular life possessed asmany as twenty SAM-binding
proteins from at least five distinct fold classes.14 Class I MTs are
predicted to have been well represented in this primordial reper-
toire of enzymes,which probably included activities for producing
SAM, synthesizing polyamines, and methylating several
substrates.14 In general, the subsequent evolutionary trajectories
of macromolecule MTs and small-molecule MTs diverged
significantly, as no single small-molecule MT appears to be
conserved across all domains of life. Therefore, most NPMTs
have apparently been ‘‘tailored to function’’ in a clade- and
sometimes species-specific manner. Abundant evidence supports
the rapid expansion of NPMT gene families in several lineages,
due to genomic duplication events.10,20,74 Concomitant or subse-
quent gene fusions probably added additional levels of substrate
recognition, at its most extreme in the emergence of entirely new
domains. Neofunctionalization following duplication ultimately
led to large families of MTs involved in many different biological
processes.20,34As such, we now find that most NPMTs participate
in so-called specializedmetabolic pathways, i.e. biosynthetic grids
restricted to certain taxa or even to individual species.
thereby promoting methyl transfer. The divalent metal ion, in this case
Ca2+, is shown as a grey sphere. The substrate caffeoyl-CoA (with its 3-O-
methyl group removed) is colored magenta, and SAH is yellow. The blue
circle labeled with a ‘W’ represents a vicinal water molecule that abstracts
the proton from the methyl acceptor atom.
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Aclacimycin 10-hydroxylase (RdmB) represents an intriguing
example of neofunctionalization of the Class I MT fold.37,75
While indications from overall structure, SAM dependence, and
similarity to DnrK80 point to an MT function for RdmB, SAM
co-substrate binding in RdmB is atypical, such that methyl
transfer cannot occur. RdmB has instead evolved an unexpected
function as a hydroxylase.37 This example emphasizes the
importance of empirical functional characterization of enzymes,
and suggests that there are likely other mis-annotations of genes
based on homology and overall structural similarity. It will be
interesting to learn of additional instances where the Class I MT
scaffold has acquired or extended its functionality to catalyze
reactions other than methylation.
Moreover, in termsof abroader viewof themolecular evolution
of the NPMTs, one can ask, does the wide diversity of sequences,
appendages to their core SAM-binding fold, and their associated
substrate specificities/promiscuities indicate that these enzymes
are more structurally and catalytically malleable and therefore
readily evolvable? The exploration of this notion will require that
we progress from single enzyme stamp collecting to more
comprehensive studies of NPMT structure-function relationships
using an exponentially expanding database of sequences, folds
and activities, tied together by consideration of the evolutionary
mechanisms and selective pressures accompanying the amazing
adaptability of the SAM-binding systems in biocatalysis.
9 Engineering of NPMTs
The diverse substrate repertoires of the NPMTs, which never-
theless share a conserved protein fold, suggests that the NPMTs
might serve as useful starting points for protein engineering to
generate new biocatalysts. However, although the potential
malleability of NPMTs is often discussed in the literature, there
are relatively few reports of the successful rational engineering of
NPMTs or the use of these enzymes in the context of metabolic
engineering. This is partly due to the difficulty associated with
SAM regeneration in a metabolic engineering context, but also,
because we have yet to go beyond a narrow focus on individual
steps in specific metabolic pathways.
The NPMTs of plant phenylpropanoid metabolism and of the
SABATH family provide a number of examples where relatively
few amino-acid substitutions in the active site cause a shift in the
substrate specificity and/or regiospecificity of methylation,81 or
an expansion of the accepted substrate range.7,77,82,83 The relaxed
substrate selectivity (or ‘promiscuity’) of some NPMTs also
makes them attractive targets for metabolic engineering.50,84,85
Regiospecific methylation in organic synthesis can be a difficult
task. Such reactions often require elaborate schemes for the
addition of protecting groups and can suffer from low yields. The
design of engineered MTs to carry out these reactions could
improve the efficiency of bioactive molecule production through
semi-synthesis or recombinant production platforms albeit
done in concert with the development of cost effective SAM
regenerating systems.
10 Conclusions and future perspectives
Unquestionably, the structural elucidation of NPMT enzymes
has significantly advanced our understanding of how these
This journal is ª The Royal Society of Chemistry 2012
enzymes function in a biological context and has provided
considerable insight into their molecular evolution. The dozens
of structure determinations of enzymes derived from all three
domains of life provide what is ostensibly a comprehensive
representation of the structural diversity of NPMTs. However, a
number of NPMT lineages have yet to be explored on the
structural level, including sterol MTs, g-tocopherol MT-related
enzymes, and the ubiE/COQ5 family. Filling in the remaining
gaps in our structure-function landscape of the NPMT super-
family will further our understanding of the structural determi-
nants governing substrate recognition and catalysis, and foster
the synthesis of a universal theory of the evolution of NPMT
structure and function.
11 Acknowledgements
J.P.N. is a Howard HughesMedical Investigator. Research in the
Noel Laboratory is supported by the Howard Hughes Medical
Institute and the National Science Foundation (MCB-0645794,
MCB-0718064 and EEC-0813570). D.K.L. is a Natural Sciences
and Engineering Research Council of Canada (NSERC) Post-
doctoral Fellow.
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