- Mechanistic Studies on CysS - A Vitamin B12-Dependent Radical SAM Methyltransferase Involved in the Biosynthesis of the tert-Butyl Group of Cystobactamid
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Cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) methyltransferases catalyze methylation reactions at non-nucleophilic centers in a wide range of substrates. CysS is a Cbl-dependent radical SAM methyltransferase involved in cystobactamid biosynthesis. This enzyme catalyzes the sequential methylation of a methoxy group to form ethoxy, i-propoxy, s-butoxy, and t-butoxy groups on a p-aminobenzoate peptidyl carrier protein thioester intermediate. This biosynthetic strategy enables the host myxobacterium to biosynthesize a combinatorial antibiotic library of 25 cystobactamid analogues. In this Article, we describe three experiments to elucidate how CysS uses Cbl, SAM, and a [4Fe-4S] cluster to catalyze iterative methylation reactions: a cyclopropylcarbinyl rearrangement was used to trap the substrate radical and to estimate the rate of the radical substitution reaction involved in the methyl transfer; a bromoethoxy analogue was used to explore the active site topography; and deuterium isotope effects on the hydrogen atom abstraction by the adenosyl radical were used to investigate the kinetic significance of the hydrogen atom abstraction. On the basis of these experiments, a revised mechanism for CysS is proposed.
- Begley, Tadhg P.,Wang, Yuanyou
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supporting information
p. 9944 - 9954
(2020/07/08)
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- Antirheumatic agents: Novel methotrexate derivatives bearing a benzoxazine or benzothiazine moiety
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Novel methotrexate (MTX) derivatives bearing dihydro-2H-1,4- benzothiazine or dihydro-2H-1,4-benzoxazine were synthesized and tested for in vitro antiproliferative activities against human synovial cells (hSC) and human peripheral blood mononuclear cells (hPBMC) obtained from patients with rheumatoid arthritis and healthy volunteers, respectively. In vivo antiarthritic activities of these derivatives were also evaluated in a rat adjuvant arthritis model. N-[[4-[(2,4-Diaminopteridin-6-yl)methyl]-3,4- dihydro-2H-1,4-benzothiazin-7-yl]carbonyl]-L-glutamic acid (3c) exhibited more potent antiproliferative activities in hSC and hPBMC than MTX in vitro. Antiproliferative activities of N-[[4-[(2,4-diaminopteridin-6-yl)methyl]- 3,4-dihydro-2H-1,4-benzoxazin-7-yl]carbonyl]-L-homoglutamic acid (3b) and N- [[4-[(2,4-diaminopteridin-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7- yl]carbonyl]-L-homoglutamic acid (3d) (MX-68) were comparable to that of MTX in these in vitro assays. Compounds 3b,d (MX-68) significantly suppressed progression of the adjuvant arthritis in a dose-dependent manner ranging from 0.5 to 2.5 mg/kg (po). In addition, 3d (MX-68) completely suppressed this progression at the dose of 2.5 mg/kg (po). Importantly, 3d (MX-68) having benzothiazine and homoglutamate, as expected, did not undergo polyglutamation, a process which may be responsible for the associated side effects of MTX. These results suggest that 3d (MX-68) is a potent and safe candidate antirheumatic agent, absent of the side effects of MTX.
- Matsuoka, Hiroharu,Ohi, Nobuhiro,Mihara, Masahiko,Suzuki, Hiroshi,Miyamoto, Katsuhito,Maruyama, Noriaki,Tsuji, Keiichiro,Kato, Nobuaki,Akimoto, Toshio,Takeda, Yasuhisa,Yano, Keiichi,Kuroki, Toshio
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p. 105 - 111
(2007/10/03)
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