Publications


Colorimetric activity measurement of a recombinant putrescine N-methyltransferase from Datura stramonium.

Biastoff S, Teuber M, Zhou ZS, Dräger B.

Planta Med. 2006 Oct;72(12):1136-41. Epub 2006 Aug 21.

Abstract

Putrescine N-methyltransferase (PMT, EC 2.1.1.53) catalyses the S-adenosyl- L-methionine (SAM or AdoMet)-dependent methylation of putrescine to N-methylputrescine within the biosynthetic pathways of calystegines, nicotine, and tropane alkaloids in medicinal plants and produces S-adenosyl- L-homocysteine (SAH or AdoHcy). Determination of PMT activity was time-consuming and hardly reproducible in the past because it required tedious separation steps after chemical derivatisation or radioactive labelling of N-methylputrescine. A convenient and accurate enzyme-coupled colorimetric assay is based on the conversion of SAH to homocysteine by 5'-methylthioadenosine/ S-adenosylhomocysteine nucleosidase (MTAN/SAHN, EC 3.2.2.9) and S-ribosylhomocysteine lyase (LuxS, EC 4.4.1.21). Homocysteine is quantified by 5,5'-dithiobis-2-nitrobenzoic acid. Putrescine was shown not to interfere with MTAN or LuxS. The colorimetric assay was validated by HPLC analysis. K(m) values determined by the assay, 108 microM for putrescine and 42 microM for SAM, are lower than the previously reported values, due to alleviation of PMT inhibition by SAH. DTNB:5,5'-dithiobis-2-nitrobenzoic acid LuxS: S-ribosylhomocysteine lyase MTAN:5'-methylthioadenosine nucleosidase PMT:putrescine N-methyltransferase SAH: S-adenosyl- L-homocysteine SAM: S-adenosyl- L-methionine TNB:2-nitro-5-thiobenzoic acid.


16924584


Carbocyclic pyrimidine nucleosides as inhibitors of S-adenosylhomocysteine hydrolase.

Mosley SL, Bakke BA, Sadler JM, Sunkara NK, Dorgan KM, Zhou ZS, Seley-Radtke KL,

Bioorg Med Chem. 2006 Dec 1;14(23):7967-71. Epub 2006 Aug 10.

Abstract

The design, synthesis, and unexpected inhibitory activity against S-adenosyl-homocysteine (SAH) hydrolase (SAHase, EC 3.3.1.1) for a series of truncated carbocyclic pyrimidine nucleoside analogues is presented. Of the four nucleosides obtained, 10 was found to be active with a Ki value of 5.0 microM against SAHase.


16904326


An enzyme-coupled continuous spectrophotometric assay for S-adenosylmethionine-dependent methyltransferases.

Dorgan KM, Wooderchak WL, Wynn DP, Karschner EL, Alfaro JF, Cui Y, Zhou ZS, Hevel JM.

Anal Biochem. 2006 Mar 15;350(2):249-55. Epub 2006 Feb 7.

Abstract

Modification of small molecules and proteins by methyltransferases affects a wide range of biological processes. Here, we report an enzyme-coupled continuous spectrophotometric assay to quantitatively characterize S-adenosyl-L-methionine (AdoMet/SAM)-dependent methyltransferase activity. In this assay, S-adenosyl-L-homocysteine (AdoHcy/SAH), the transmethylation product of AdoMet-dependent methyltransferases, is hydrolyzed to S-ribosylhomocysteine and adenine by recombinant S-adenosylhomocysteine/5'-methylthioadenosine nucleosidase (SAHN/MTAN, EC 3.2.2.9). Subsequently, adenine generated from AdoHcy is further hydrolyzed to hypoxanthine and ammonia by recombinant adenine deaminase (EC 3.5.4.2). This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Coupling enzymes are recombinant and easily purified. The utility of this assay was shown using recombinant rat protein arginine N-methyltransferase 1 (PRMT1, EC 2.1.1.125), which catalyzes the mono- and dimethylation of guanidino nitrogens of arginine residues in select proteins. Using this assay, the kinetic parameters of PRMT1 with three synthetic peptides were determined. An advantage of this assay is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S-adenosylmethionine-dependent methyltransferases. Finally, this method may be used to assay other enzymes that produce AdoHcy, 5'-methylthioadenosine, or compounds that can be cleaved by AdoHcy nucleosidase.


16460659


Potential Beneficial Metabolic Interactions Between Tamoxifen and Isoflavones via Cytochrome P450-mediated Pathways in Female Rat Liver Microsomes

Jun Chen, Steven C. Halls, Joshua F. Alfaro, Zhaohui Zhou, Ming Hu.

Pharmaceutical Research November 2004, Volume 21, Issue 11, pp 2095-2104

Abstract

Purpose. This study aims to evaluate a cytochrome P450-based tamoxifen-isoflavone interaction and to determine the mechanisms responsible for inhibitory effects of isoflavones (e.g., genistein) on the formation of α-hydroxytamoxifen.

Methods. Metabolism studies were performed in vitro using female rat liver microsomes. The effects of genistein and an isoflavone mixture on tamoxifen metabolism and the inhibition mechanism were determined using standard kinetic analysis, preincubation, and selective chemical inhibitors of P450.

Results. Metabolism of tamoxifen was saturable with K m values of 4.9 ± 0.6, 14.6 ± 2.2, 25 ± 5.9 μM and V max values of 34.7 ± 1.4, 297.5 ± 19.2, 1867 ± 231 pmol min−1 mg−1 for α-hydroxylation, N-desmethylation, and N-oxidation, respectively. Genistein (25 μM) inhibited α-hydroxylation at 2.5 μM tamoxifen by 64% (p < 0.001) but did not affect the 4-hydroxylation, N-desmethylation, and N-oxidation. A combination of three (genistein, daidzein, and glycitein) to five isoflavones (plus biochanin A and formononetin) inhibited tamoxifen α-hydroxylation to a greater extent but did not decrease the formation of identified metabolites. The inhibition on α-hydroxylation by genistein was mixed-typed with a K i , value of 10.6 μM. Studies using selective chemical inhibitors showed that tamoxifen α-hydroxylation was mainly mediated by rat CYP1A2 and CYP3A1/2 and that genistein 3`-hydroxylation was mainly mediated by rat CYP1A2, CYP2C6 and CYP2D1.

Conclusions. Genistein and its isoflavone analogs have the potential to decrease side effects of tamoxifen through metabolic interactions that inhibit the formation of α-hydroxytamoxifen via inhibition of CYP1A2.


15587933


Synthesis of LuxS inhibitors targeting bacterial cell-cell communication.

Alfaro JF, Zhang T, Wynn DP, Karschner EL, Zhou ZS.

Org Lett. 2004 Sep 2;6(18):3043-6.

Abstract

[reaction: see text] Quorum sensing is a process by which bacteria sense cell density. This cell-cell communication process is mediated by autoinducers. A cross-species messenger, autoinducer-2 (AI-2) is produced from S-ribosyl-L-homocysteine by the LuxS enzyme. A proposed mechanism for LuxS is an aldose-ketose isomerization of S-ribosylhomocysteine followed by a beta-elimination. We report here the synthesis of two substrate analogues, S-anhydroribosyl-L-homocysteine and S-homoribosyl-L-cysteine, which prevent the initial and final step of the mechanism, respectively.


15330583


An enzyme-coupled colorimetric assay for S-adenosylmethionine-dependent methyltransferases.

Hendricks CL, Ross JR, Pichersky E, Noel JP, Zhou ZS.

Anal Biochem. 2004 Mar 1;326(1):100-5.

Abstract

We report here an enzyme-coupled colorimetric assay for salicylic acid carboxyl methyltransferase (SAMT), which utilizes S-adenosyl-l-methionine (AdoMet or SAM) as the methyl donor. In this assay, S-adenosyl-l-homocysteine (AdoHcy or SAH), a common product of AdoMet-dependent transmethylation reactions, is first hydrolyzed by recombinant AdoHcy nucleosidase (EC 3.2.2.9) into adenine and S-ribosylhomocysteine. Recombinant LuxS (S-ribosylhomocysteinase, EC 3.2.1.148) cleaves the latter compound to form homocysteine. Finally, homocysteine is quantified using Ellman's reagent and the accompanying absorption change at 412nm through recording using a microplate format. Notably, SAMT and most AdoMet-dependent methyltransferases undergo marked AdoHcy-mediated product inhibition. As such, an additional advantage of this assay which includes AdoHcy nucleosidase is the destruction of AdoHcy, thus alleviating product inhibition. Under our assay conditions, complete substrate conversion is observed and precise kinetic parameters can be determined in a facile and quantitative manner. This assay should be generally applicable to other AdoMet-dependent methyltransferases. Moreover, the procedure is easily amendable to batch assay and high-throughput screening approaches.


14769341


Rapid screening for S-adenosylmethionine-dependent methylation products by enzyme-transferred isotope patterns analysis.

Wan W, Zhao G, Al-Saad K, Siems WF, Zhou ZS.

Rapid Commun Mass Spectrom. 2004;18(3):319-24.

Abstract

We report here an isotopic labeling and mass spectrometric method to rapidly identify S-adenosylmethionine (AdoMet)-dependent methylation products. In the presence of CH(3)- and CD(3)-labeled AdoMet, a methyl transfer product appears as a doublet separated by 3 Da in a mass spectrum, while other compounds show their normal isotopic distribution. Based on this unique isotopic pattern, methylation product(s) can be easily detected even from a mixture of cellular components. To validate our method, the product of human thiopurine methyltransferase (TPMT, EC 2.1.1.67) has been successfully identified from both an in vitro assay and a whole-cell assay. This method is generally applicable to AdoMet-dependent transmethylation and other group-transfer reactions, and constitutes the first example of a general strategy of enzyme-transferred isotope patterns (ETIPs) analysis.


14755618


Cobalamin-Dependent and Cobalamin-Independent Methionine Synthases: Are There Two Solutions to the Same Chemical Problem?

Rowena G. Matthews, April E. Smith, Zhaohui S. Zhou, Rebecca E. Taurog, Vahe Bandarian, John C. Evans, Martha Ludwig.

Helvetica Chimica Acta, Volume 86, Issue 12, pages 3939–3954, December 2003
DOI: 10.1002/hlca.200390329

Abstract

Two enzymes in Escherichia coli, cobalamin-independent methionine synthase (MetE) and cobalamin-dependent methionine synthase (MetH), catalyze the conversion of homocysteine (Hcy) to methionine using N(5)-methyltetrahydrofolate (CH3-H4folate) as the Me donor. Despite the absence of sequence homology, these enzymes employ very similar catalytic strategies. In each case, the pKa for the SH group of Hcy is lowered by coordination to Zn2+, which increases the concentration of the reactive thiolate at neutral pH. In each case, activation of CH3-H4folate appears to involve protonation at N(5). CH3-H4folate remains unprotonated in binary E⋅CH3-H4folate complexes, and protonation occurs only in the ternary E⋅CH3-H4folate⋅Hcy complex in MetE, or in the ternary E⋅CH3-H4folate⋅cob(I)alamin complex in MetH. Surprisingly, the similarities are proposed to extend to the structures of these two unrelated enzymes. The structure of a homologue of the Hcy-binding region of MetH, betainehomocysteine methyltransferase, has been determined. A search of the three-dimensional-structure data base by means of the structure-comparison program DALI indicates similarity of the BHMT structure with that of uroporphyrin decarboxylase (UroD), a homologue of the MT2-A and MT2-M proteins from Archaea, which catalyze Me transfers from methylcorrinoids to coenzyme M and share the Zn-binding scaffold of MetE. Here, we present a model for the Zn binding site of MetE, obtained by grafting the Zn ligands of MT2-A onto the structure of UroD.


8304207


Chemical synthesis of S-ribosyl-L-homocysteine and activity assay as a LuxS substrate.

Zhao G, Wan W, Mansouri S, Alfaro JF, Bassler BL, Cornell KA, Zhou ZS.

Bioorg Med Chem Lett. 2003 Nov 17;13(22):3897-900.

Abstract

Bacterial quorum sensing is mediated by autoinducers, small signaling molecules generated by bacteria. It has been proposed that the LuxS enzyme converts S-ribosyl-L-homocysteine to 4,5-dihydroxy-2,3-pentanedione, the precursor of autoinducer 2 (AI-2). We report here a chemical synthesis of S-ribosyl-L-homocysteine and its analogue using Mitsunobu coupling. Chemically synthesized ribosylhomocysteine has been confirmed as a substrate for LuxS in both an enzyme assay and a whole cell quorum sensing assay. The chemical entities of products from the LuxS reaction were also established. Several ribosylhomocysteine analogues have been tested as LuxS inhibitors.


14592470


A stereospecific colorimetric assay for (S,S)-adenosylmethionine quantification based on thiopurine methyltransferase-catalyzed thiol methylation.

Cannon LM, Butler FN, Wan W, Zhou ZS.

Anal Biochem. 2002 Sep 15;308(2):358-63.

Abstract

S-Adenosyl-L-methionine (AdoMet) which is biologically synthesized by AdoMet synthetase bears an S configuration at the sulfur atom. The chiral sulfonium spontaneously racemizes to form a mixture of S and R isomers of AdoMet under physiological conditions or normal storage conditions. The chirality of AdoMet greatly affects its activity; the R isomer is not accepted as a substrate for AdoMet-dependent methyltransferases. We report a stereospecific colorimetric assay for (S,S)-adenosylmethionine quantification based on an enzyme-coupled reaction in which (S,S)-AdoMet reacts with 2-nitro-5-thiobenzoic acid to form AdoHcy and 2-nitro-5-methylthiobenzoic acid. The transformation is catalyzed by recombinant human thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) and is associated with a large spectral change at 410 nm. Accumulation of the S-adenosylhomocysteine (AdoHcy) product, a feedback inhibitor of TPMT, slows the assay. AdoHcy nucleosidase (EC 3.2.2.9) irreversibly cleaves AdoHcy to adenine and S-ribosylhomocysteine, significantly shortening the assay time to less than 10 min. The assay is linear from 5 to at least 60 microM (S,S)-AdoMet.


12419350


Vinyl sulfonium as novel proteolytic enzyme inhibitor.

Zhao G, Zhou ZS,

Bioorg Med Chem Lett. 2001 Sep 3;11(17):2331-5.

Abstract

Vinyl sulfoniums were synthesized from vinyl sulfides by methylation, and inhibited the proteolytic enzyme papain. Inhibition studies suggest a mechanism by which the vinyl sulfonium inhibitor covalently and irreversibly modifies the enzyme.


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