Crystal structures of pristine and oxidatively processed lignin peroxidase expressed in Escherichia coli and of the W171F variant that eliminates the redox active tryptophan 171.: Implications for the reaction mechanism

被引:84
作者
Blodig, W
Smith, AT
Doyle, WA
Piontek, K
机构
[1] Swiss Fed Inst Technol, Inst Biochem, CH-8092 Zurich, Switzerland
[2] Univ Sussex, Sch Biol Sci, Brighton BN1 9QG, E Sussex, England
关键词
lignin peroxidase; catalytic mechanism; crystal structure; redox cycle; protein radical;
D O I
10.1006/jmbi.2000.4346
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The heme enzyme lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium contains a solvent exposed redox active tryptophan residue (Trp171) that carries a unique hydroxy group stereospecifically attached to its C-beta atom. A Trp171Phe mutant has no activity at all towards the substrate veratryl alcohol. The mechanism of veratryl alcohol oxidation involving beta -hydroxy-Trp171 is largely unknown. Here, we present the first crystal structures of LiP isozyme H8 at high resolution in its pristine non-hydroxylated form, of the C-beta-hydroxylated form, and of the Trp171Phe mutant using recombinantly expressed and refolded protein produced from Escherichia coli. As a consequence, all structures are unglycosylated. Structural changes in response to the mutation are marginal and allow us to attribute the complete lack of activity exclusively to the absence of the redox active indole side-chain. The origin of the stereospecificity of the Trp171 hydroxylation can be explained on structural grounds. A reaction mechanism involving Trp171 is proposed and the possible function of the modification is discussed. Another important result regarding the ongoing debate on the co-ordination state of the heme iron in the resting state is that the iron is six coordinate in all cases the data being collected at room temperature. The mean distance from the iron to the distal water Ligand is 2.18(+/-0.08) Angstrom. The radical scavenger orcinol was found to decrease radiation damage to the crystals, during data collection at room temperature. (C) 2001 Academic Press.
引用
收藏
页码:851 / 861
页数:11
相关论文
共 38 条
  • [1] LIGNIN PEROXIDASE - RESONANCE RAMAN SPECTRAL EVIDENCE FOR COMPOUND-II AND FOR A TEMPERATURE-DEPENDENT COORDINATION-STATE EQUILIBRIUM IN THE FERRIC ENZYME
    ANDERSSON, LA
    RENGANATHAN, V
    LOEHR, TM
    GOLD, MH
    [J]. BIOCHEMISTRY, 1987, 26 (08) : 2258 - 2263
  • [2] THE CCP4 SUITE - PROGRAMS FOR PROTEIN CRYSTALLOGRAPHY
    BAILEY, S
    [J]. ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 1994, 50 : 760 - 763
  • [3] Lifetime, reduction potential and base-induced fragmentation of the veratryl alcohol radical cation in aqueous solution. Pulse radiolysis studies on a ligninase "mediator"
    Bietti, M
    Baciocchi, E
    Steenken, S
    [J]. JOURNAL OF PHYSICAL CHEMISTRY A, 1998, 102 (38) : 7337 - 7342
  • [4] Evidence from spin-trapping for a transient radical on tryptophan residue 171 of lignin peroxidase
    Blodig, W
    Smith, AT
    Winterhalter, K
    Piontek, K
    [J]. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1999, 370 (01) : 86 - 92
  • [5] Autocatalytic formation of a hydroxy group at Cβ of Trp171 in lignin peroxidase
    Blodig, W
    Doyle, WA
    Smith, AT
    Winterhalter, K
    Choinowski, T
    Piontek, K
    [J]. BIOCHEMISTRY, 1998, 37 (25) : 8832 - 8838
  • [6] BRUNGER AT, 1992, XPLOR VERSION 3 1 SY
  • [7] Description of a versatile peroxidase involved in the natural degradation of lignin that has both manganese peroxidase and lignin peroxidase substrate interaction sites
    Camarero, S
    Sarkar, S
    Ruiz-Dueñas, FJ
    Martínez, MJ
    Martínez, AT
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (15) : 10324 - 10330
  • [8] The crystal structure of lignin peroxidase at 1.70 Å resolution reveals a hydroxy group on the Cβ of tryptophan 171:: A novel radical site formed during the redox cycle
    Choinowski, T
    Blodig, W
    Winterhalter, KH
    Piontek, K
    [J]. JOURNAL OF MOLECULAR BIOLOGY, 1999, 286 (03) : 809 - 827
  • [9] CHOINOWSKI T, 1996, THESIS ETH ZURICH
  • [10] ANALYSIS OF NUCLEOTIDE-SEQUENCES OF 2 LIGNINASE CDNAS FROM A WHITE-ROT FILAMENTOUS FUNGUS, PHANEROCHAETE-CHRYSOSPORIUM
    DEBOER, HA
    ZHANG, YZ
    COLLINS, C
    REDDY, CA
    [J]. GENE, 1987, 60 (01) : 93 - 102