메뉴 건너뛰기




Volumn 126, Issue 23, 2004, Pages 7212-7221

Aristolochene synthase: Mechanistic analysis of active site residues by site-directed mutagenesis

Author keywords

[No Author keywords available]

Indexed keywords

AMINO ACIDS; MUTAGENESIS; PHOSPHATES; REDUCTION;

EID: 2942558483     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja0499593     Document Type: Article
Times cited : (93)

References (71)
  • 10
    • 0000558630 scopus 로고
    • (b) Cane, D. E. Chem. Rev. 1990, 90, 1089-1103.
    • (1990) Chem. Rev. , vol.90 , pp. 1089-1103
    • Cane, D.E.1
  • 11
    • 0001182551 scopus 로고    scopus 로고
    • Cane, D. E., Ed. (Volume 2 in Comprehensive Natural Products Chemistry; Barton, D., Nakanishi, K., Meth-Cohn, O., Eds.); Pergamon Press: Oxford
    • (c) Cane, D. E. In Isoprenoids Including Carotenoid and Steroids; Cane, D. E., Ed. (Volume 2 in Comprehensive Natural Products Chemistry; Barton, D., Nakanishi, K., Meth-Cohn, O., Eds.); Pergamon Press: Oxford, 1999; pp 155-215.
    • (1999) Isoprenoids Including Carotenoid and Steroids , pp. 155-215
    • Cane, D.E.1
  • 46
    • 2142697059 scopus 로고    scopus 로고
    • Cane, D. E., Ed. (Volume 2 in Comprehensive Natural Products Chemistry; Barton, D., Nakanishi, K., Meth-Cohn, O., Eds.); Pergamon Press: Oxford
    • (a) Croteau, R. B. In Isoprenoids Including Carotenoids and Steroids; Cane, D. E., Ed. (Volume 2 in Comprehensive Natural Products Chemistry; Barton, D., Nakanishi, K., Meth-Cohn, O., Eds.); Pergamon Press: Oxford, 1999; pp 97-153.
    • (1999) Isoprenoids Including Carotenoids and Steroids , pp. 97-153
    • Croteau, R.B.1
  • 56
    • 2942551763 scopus 로고    scopus 로고
    • note
    • As previously reported, the activity of P. roqueforti aristolochene synthase is a simple linear function of protein concentration only within a specific concentration range.10 The activity of wild-type aristolochene synthase was linearly proportional to protein concentration only up to 20 nM, after which the measured activity per mg protein decreased, with negligible increase in total activity above 60 nM protein (Figure 2A, Table 1). Similar behavior was also observed for the A. terreus aristolochene synthase, which displayed nonlinear dependence on protein concentration above 27 nM protein. To allow meaningful comparisons of steady state kinetic parameters, therefore, we first determined the concentration dependence of aristolochene synthase activity for all mutants prepared in this study. (Table 1) Kinetic assays of each mutant were then carried out only at protein concentrations within the experimentally established linear range.
  • 57
    • 2942603802 scopus 로고    scopus 로고
    • note
    • The activity of the Y92F mutant was found to be a simple linear function of protein concentration only up to 50 nM (Figure 2B, Table 1), with only minor increases in total cyclase activity above 80 nM mutant protein.
  • 61
    • 2942608489 scopus 로고    scopus 로고
    • note
    • m compared to the wild-type. The reported steady-state kinetic parameters were unfortunately determined at concentrations well above what turns out to be the maximum 50 nM protein concentration for linear dependence of Y92F activity (cf Table 1), thereby compromising their quantitative significance. On the basis of an observed increase in the proportion of germacrene A (28%), of unspecified configuration, the implausible suggestion was made that Tyr-92 must be the normal active site Lewis acid responsible for the cyclization of the germacrene A intermediate, although the major product isolated was still aristolochene. These workers also reported the isolation of the coproducts α-selinene (6), β-selinene, and selina-4,11-diene, none of which are detectable in our analysis of the same Y92F mutant (Table 2). The latter compounds are well-known artifacts of the acid-catalyzed rearrangement of germacrene A. Rearrangement of germacrene A is easily avoided by chromatography over aluminum oxide instead of silica gel and the use of sodium sulfate in place of magnesium sulfate as drying agent.
  • 66
  • 67
    • 0004270170 scopus 로고    scopus 로고
    • Wiley: New York
    • Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning: a Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989. Current Protocols in Molecular Biology, CD-ROM; Wiley: New York, 1999.
    • (1999) Current Protocols in Molecular Biology, CD-ROM
  • 69
    • 0042622380 scopus 로고    scopus 로고
    • Schwede, T.; Kopp, J.; Guex, N.; Peitsch, M. C. Nucleic Acids Res. 2003, 31, 3381-3385. Guex, N.; Peitsch, M. C. Electrophoresis 1997, 18, 2714-2723. Peitsch, M. C. Bio/Technology 1995, 13, 658-660.
    • (2003) Nucleic Acids Res. , vol.31 , pp. 3381-3385
    • Schwede, T.1    Kopp, J.2    Guex, N.3    Peitsch, M.C.4
  • 70
    • 0031473847 scopus 로고    scopus 로고
    • Schwede, T.; Kopp, J.; Guex, N.; Peitsch, M. C. Nucleic Acids Res. 2003, 31, 3381-3385. Guex, N.; Peitsch, M. C. Electrophoresis 1997, 18, 2714-2723. Peitsch, M. C. Bio/Technology 1995, 13, 658-660.
    • (1997) Electrophoresis , vol.18 , pp. 2714-2723
    • Guex, N.1    Peitsch, M.C.2
  • 71
    • 0029004590 scopus 로고
    • Schwede, T.; Kopp, J.; Guex, N.; Peitsch, M. C. Nucleic Acids Res. 2003, 31, 3381-3385. Guex, N.; Peitsch, M. C. Electrophoresis 1997, 18, 2714-2723. Peitsch, M. C. Bio/Technology 1995, 13, 658-660.
    • (1995) Bio/Technology , vol.13 , pp. 658-660
    • Peitsch, M.C.1


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.