S-Adenosyl-L-methionine-dependent macromolecule methyltransferases: potential targets for the design of chemotherapeutic agents

Borchardt, Ronald T.

doi:10.1021/jm00178a001

Cited by 161 publications

(83 citation statements)

References 28 publications

(33 reference statements)

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“…Dibasic anhydrous sodium phosphate (Na 2 HPO 4 ) was obtained from Mallinckrodt (Hazelwood, MO, USA). Monobasic monohydrate sodium phosphate (NaH 2 PO 4 Competitive FP assay for methyltransferase activity COMT endpoint methyltransferase assays were performed using an enzyme reaction volume of 30 μl followed by the addition of 20 μl anti-AdoHcy antibody/tracer complex. COMT enzyme reactions used 0 to 0.33 μg COMT enzyme as indicated, 250 μM DBA, and 3 μM AdoMet in COMT assay buffer (20 mM sodium phosphate [pH 7.5], 5 mM MgCl 2 , and 1 mM DTT).…”

Section: Methodsmentioning

confidence: 99%

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A universal competitive fluorescence polarization activity assay for S-adenosylmethionine utilizing methyltransferases

Graves

Zhang

Scott

2008

Analytical Biochemistry

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A high-throughput, competitive fluorescence polarization immunoassay has been developed for the detection of methyltransferase activity. The assay was designed to detect Sadenosylhomocysteine (AdoHcy), a product of all S-adenosylmethionine (AdoMet)-utilizing methyltransferase reactions. We employed commercially available anti-AdoHcy antibody and fluorescein-AdoHcy conjugate tracer to measure AdoHcy generated as a result of methyltransferase activity. AdoHcy competes with tracer in the antibody/tracer complex. The release of tracer results in a decrease in fluorescence polarization. Under optimized conditions, AdoHcy and AdoMet titrations demonstrated that the antibody had more than a 150-fold preference for binding AdoHcy relative to AdoMet. Mock methyltransferase reactions using both AdoHcy and AdoMet indicated that the assay tolerated 1 to 3 μM AdoMet. The limit of detection was approximately 5 nM (0.15 pmol) AdoHcy in the presence of 3 μM AdoMet. To validate the assay's ability to quantitate methyltransferase activity, the methyltransferase catechol-Omethyltransferase (COMT) and a known selective inhibitor of COMT activity were used in proofof-principle experiments. A time-and enzyme concentration-dependent decrease in fluorescence polarization was observed in the COMT assay that was developed. The IC 50 value obtained using a selective COMT inhibitor was consistent with previously published data. Thus, this sensitive and homogeneous assay is amenable for screening compounds for inhibitors of methyltransferase activity. KeywordsFluorescence polarization; Methyltransferase; S-Adenosylhomocysteine; S-Adenosylmethionine; Catechol-O-methyltransferase Methyltransferases are a diverse class of enzymes that catalyze the transfer of a single methyl group from a methyl donor molecule to a methyl acceptor. The vast majority use Sadenosylmethionine (AdoMet) 1 as the methyl donor [1,2]. AdoMet is the second most HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscriptwidely used substrate molecule in the cell after ATP. Methylation is an essential and highly choreographed regulatory metabolic process. Small organic molecules and peptides and most types of cellular macromolecules, including DNA, RNA, protein, and lipids, can serve as substrates for methylation (for detailed reviews, see Refs. [2][3][4][5][6][7]).A rapidly growing area of methyltransferase research focuses on methylation of histone tails by histone methyltransferases (HMTs) (for reviews, see Refs. [8,9]). Histone methylation is an epigenetic modification that has a role in formation of heterochromatin, X chromosome inactivation, and modification of gene expression patterns. Histone methylation occurs on arginine and lysine residues and is catalyzed by methyltransferases belonging to three distinct families of proteins: the protein arginine N-methyltransferase 1 (PRMT1) family, the SET-domain-containing protein family, and the non-SET-domain proteins DOT1/DOT1L. This histone methylation alters chromatin structure and, thus, regulates...

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Section: Methodsmentioning

confidence: 99%

“…Small organic molecules and peptides and most types of cellular macromolecules, including DNA, RNA, protein, and lipids, can serve as substrates for methylation (for detailed reviews, see Refs. [2][3][4][5][6][7]). …”

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A universal competitive fluorescence polarization activity assay for S-adenosylmethionine utilizing methyltransferases

Graves

Zhang

Scott

2008

Analytical Biochemistry

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“…22 In addition to quorum sensing, Pfs is involved in important cellular processes, such as protein and DNA methylation and metabolism and polyamine biosynthesis. [23][24][25][26] The involvement of Pfs in important bacterial processes makes it an appealing target for the design of new antibiotics. Importantly for antibiotic design, mammalian cells, in contrast, utilize the enzyme SAH hydrolase, which cleaves SAH into homocysteine and adenosine.…”

Section: Introductionmentioning

confidence: 99%

Towards area‐based in vitro metabolic engineering: Assembly of Pfs enzyme onto patterned microfabricated chips

Lewandowski

Bentley

et al. 2008

Biotechnology Progress

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in Wiley InterScience (www.interscience.wiley.com).We report an approach for spatially selective assembly of an enzyme onto selected patterns of microfabricated chips. Our approach is based on electrodeposition of the aminopolysaccharide chitosan onto selected electrode patterns and covalent conjugation of a target enzyme to chitosan upon biochemical activation of a genetically fused ''pro-tag.'' We report assembly of S-adenosylhomocysteine nucleosidase (Pfs) fused with a C-terminal pentatyrosine pro-tag. Pfs is a member of the bacterial autoinducer-2 biosynthesis pathway, catalyzing the irreversible cleavage of S-adenosylhomocysteine. The assembled Pfs retains its catalytic activity and structure, as demonstrated by retained antibody recognition. Assembly is controlled by the electrode area, resulting in reproducible rates of catalytic conversion for a given area, and thus allowing for area-based manipulation of catalysis and small molecule biosynthesis. Our approach enables optimization of small molecule biosynthesis in 1-step as well as multistep enzymatic reactions, including entire metabolic pathways, and we envision a wide variety of potential applications.

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“…MTAN is involved in bacterial pathways related to polyamine biosynthesis, quorum sensing, methyl transfer reactions and adenine and methionine salvage ( Figure 1;3-6). The substrates of MTAN are structurally related to S-adenosylmethionine (SAM) and function as product inhibitors in SAM-requiring reactions (7). In bacteria, accumulation of these metabolites is avoided through the function of the pfs gene.…”

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confidence: 99%

Structure and Inhibition of a Quorum Sensing Target from Streptococcus pneumoniae

et al. 2006

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Streptococcus pneumoniae 5′-methylthioadenosine/S-adenosylhomocysteine hydrolase (MTAN) catalyzes the hydrolytic deadenylation of its substrates to form adenine and 5-methylthioribose or S-ribosylhomocysteine (SRH). MTAN is not found in mammals but is involved in bacterial quorum sensing. MTAN gene disruption affects growth and pathogenicity of bacteria, making it a target for antibiotic design. Kinetic isotope effects and computational studies have established a dissociative SN1 transition state for E. coli MTAN and transition state analogues resembling the transition state are powerful inhibitors of the enzyme (Singh, V., Lee, J. L., Núñez, S., Howell, P. L. and Schramm, V. L. (2005) Biochemistry 44, 11647-11659). The MTAN from S. pneumoniae has 40% sequence identity to E. coli MTAN, but exhibits remarkably distinct kinetic and inhibitory properties. 5′-Methylthio-Immucillin-A (MT-ImmA) is a transition state analogue resembling an early SN1 transition state. It is a weak inhibitor of S. pneumoniae MTAN with a K i of 1.0 μM. The X-ray structure of S. pneumoniae MTAN with MT-ImmA indicates a dimer with the methylthio group in a flexible hydrophobic pocket. Replacing the methyl group with phenyl (PhT-ImmA), tolyl (p-TolTImmA) or ethyl (EtT-ImmA) groups increases the affinity to give K i values of 335 nM, 60 nM and 40 nM, respectively. DADMe-Immucillins are geometric and electrostatic mimics of a fullydissociated transition state and bind more tightly than Immucillins. MT-DADMe-Immucillin-A inhibits with a K i value of 24 nM and replacing the 5′-methyl group with p-Cl-phenyl (p-Cl-PhTDADMe-ImmA) gave a K i * value of 0.36 nM. The inhibitory potential of DADMe-Immucillins relative to the Immucillins supports a fully dissociated transition state structure for S. pneumoniae MTAN. Comparison of active site contacts in the X-ray crystal structures of E. coli and S. pneumoniae MTAN with MT-ImmA would predict equal binding, yet most analogues bind 10 3 to 10 4 fold more tightly to the E. coli enzyme. Catalytic site efficiency is primarily responsible for this difference since k cat /K m for S. pneumoniae MTAN is <10 -2 that of E. coli MTAN. Keywords5′-methylthioadenosine; 5′-methylthioadenosine nucleosidase; quorum sensing; nucleoside hydrolase; transition state; transition state analogue inhibitors; polyamines; MTAN structure; catalytic efficiency *Corresponding author: telephone (718) 430-2813, Fax (718) Email vern@aecom.yu.edu. 3 Immucillin-H is (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol and has been shown to have a pK a > 10 at N7 (47). MT-ImmA is chemically similar in the 9-deazaadenine ring and is expected to have similar pK a . NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript5′-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN 1 ) is a bacterial enzyme encoded by the pfs gene and catalyzes hydrolytic depurination of 5′-methylthioadenosine (MTA) to form 5-methylthioribose (MTR) and S-adenosylhomocysteine (SAH) to S-ribosylh...

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S-Adenosyl-L-methionine-dependent macromolecule methyltransferases: potential targets for the design of chemotherapeutic agents

Cited by 161 publications

References 28 publications

A universal competitive fluorescence polarization activity assay for S-adenosylmethionine utilizing methyltransferases

A universal competitive fluorescence polarization activity assay for S-adenosylmethionine utilizing methyltransferases

Towards area‐based in vitro metabolic engineering: Assembly of Pfs enzyme onto patterned microfabricated chips

Structure and Inhibition of a Quorum Sensing Target from Streptococcus pneumoniae

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