3WVS

Crystal Structure of Cytochrome P450revI


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.40 Å
  • R-Value Free: 0.188 
  • R-Value Work: 0.173 
  • R-Value Observed: 0.174 

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Ligand Structure Quality Assessment 


This is version 1.3 of the entry. See complete history


Literature

Structure-function analyses of cytochrome P450revI involved in reveromycin A biosynthesis and evaluation of the biological activity of its substrate, reveromycin T.

Takahashi, S.Nagano, S.Nogawa, T.Kanoh, N.Uramoto, M.Kawatani, M.Shimizu, T.Miyazawa, T.Shiro, Y.Osada, H.

(2014) J Biol Chem 289: 32446-32458

  • DOI: https://doi.org/10.1074/jbc.M114.598391
  • Primary Citation of Related Structures:  
    3WVS

  • PubMed Abstract: 

    Numerous cytochrome P450s are involved in secondary metabolite biosynthesis. The biosynthetic gene cluster for reveromycin A (RM-A), which is a promising lead compound with anti-osteoclastic activity, also includes a P450 gene, revI. To understand the roles of P450revI, we comprehensively characterized the enzyme by genetic, kinetic, and structural studies. The revI gene disruptants (ΔrevI) resulted in accumulation of reveromycin T (RM-T), and revI gene complementation restored RM-A production, indicating that the physiological substrate of P450revI is RM-T. Indeed, the purified P450revI catalyzed the C18-hydroxylation of RM-T more efficiently than the other RM derivatives tested. Moreover, the 1.4 Å resolution co-crystal structure of P450revI with RM-T revealed that the substrate binds the enzyme with a folded compact conformation for C18-hydroxylation. To address the structure-enzyme activity relationship, site-directed mutagenesis was performed in P450revI. R190A and R81A mutations, which abolished salt bridge formation with C1 and C24 carboxyl groups of RM-T, respectively, resulted in significant loss of enzyme activity. The interaction between Arg(190) and the C1 carboxyl group of RM-T elucidated why P450revI was unable to catalyze both RM-T 1-methyl ester and RM-T 1-ethyl ester. Moreover, the accumulation of RM-T in ΔrevI mutants enabled us to characterize its biological activity. Our results show that RM-T had stronger anticancer activity and isoleucyl-tRNA synthetase inhibition than RM-A. However, RM-T showed much less anti-osteoclastic activity than RM-A, indicating that hemisuccinate moiety is important for the activity. Structure-based P450revI engineering for novel hydroxylation and subsequent hemisuccinylation will help facilitate the development of RM derivatives with anti-osteoclast activity.


  • Organizational Affiliation

    From the Chemical Biology Group, RIKEN Center for Sustainable Resource Science, Saitama 351-0198, Japan, the Antibiotics Laboratory, RIKEN, Saitama 351-0198, Japan, shunjitaka@riken.jp.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Putative monooxygenase401Streptomyces sp. SN-593Mutation(s): 0 
Gene Names: revI
UniProt
Find proteins for G1UDU7 (Streptomyces sp. SN-593)
Explore G1UDU7 
Go to UniProtKB:  G1UDU7
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupG1UDU7
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 4 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
HEM
Query on HEM

Download Ideal Coordinates CCD File 
B [auth A]PROTOPORPHYRIN IX CONTAINING FE
C34 H32 Fe N4 O4
KABFMIBPWCXCRK-RGGAHWMASA-L
RRM
Query on RRM

Download Ideal Coordinates CCD File 
C [auth A](2E,4S,5S,6E,8E)-10-{(2R,3S,6S,8R,9S)-9-butyl-8-[(1E,3E)-4-carboxy-3-methylbuta-1,3-dien-1-yl]-3-methyl-1,7-dioxaspiro[5.5]undec-2-yl}-5-hydroxy-4,8-dimethyldeca-2,6,8-trienoic acid
C32 H48 O7
YEZCSKCHOPBNCC-OXNFKLAGSA-N
TLA
Query on TLA

Download Ideal Coordinates CCD File 
D [auth A]L(+)-TARTARIC ACID
C4 H6 O6
FEWJPZIEWOKRBE-JCYAYHJZSA-N
GOL
Query on GOL

Download Ideal Coordinates CCD File 
E [auth A],
F [auth A],
G [auth A],
H [auth A],
I [auth A]
GLYCEROL
C3 H8 O3
PEDCQBHIVMGVHV-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.40 Å
  • R-Value Free: 0.188 
  • R-Value Work: 0.173 
  • R-Value Observed: 0.174 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 52.779α = 90
b = 73.472β = 112.11
c = 58.305γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
SOLVEphasing
RESOLVEphasing
REFMACrefinement
PDB_EXTRACTdata extraction
HKL-2000data collection
HKL-2000data reduction
HKL-2000data scaling

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2014-10-01
    Type: Initial release
  • Version 1.1: 2014-10-08
    Changes: Database references
  • Version 1.2: 2017-11-22
    Changes: Refinement description
  • Version 1.3: 2022-08-24
    Changes: Database references, Derived calculations