3A6J

E122Q mutant creatininase complexed with creatine


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.00 Å
  • R-Value Free: 0.212 
  • R-Value Work: 0.195 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Substitution of Glu122 by glutamine revealed the function of the second water molecule as a proton donor in the binuclear metal enzyme creatininase

Yamashita, K.Nakajima, Y.Matsushita, H.Nishiya, Y.Yamazawa, R.Wu, Y.F.Matsubara, F.Oyama, H.Ito, K.Yoshimoto, T.

(2010) J Mol Biol 396: 1081-1096

  • DOI: https://doi.org/10.1016/j.jmb.2009.12.045
  • Primary Citation of Related Structures:  
    3A6D, 3A6E, 3A6F, 3A6G, 3A6H, 3A6J, 3A6K, 3A6L

  • PubMed Abstract: 

    Creatininase is a binuclear zinc enzyme and catalyzes the reversible conversion of creatinine to creatine. It exhibits an open-closed conformational change upon substrate binding, and the differences in the conformations of Tyr121, Trp154, and the loop region containing Trp174 were evident in the enzyme-creatine complex when compared to those in the ligand-free enzyme. We have determined the crystal structure of the enzyme complexed with a 1-methylguanidine. All subunits in the complex existed as the closed form, and the binding mode of creatinine was estimated. Site-directed mutagenesis revealed that the hydrophobic residues that show conformational change upon substrate binding are important for the enzyme activity. We propose a catalytic mechanism of creatininase in which two water molecules have significant roles. The first molecule is a hydroxide ion (Wat1) that is bound as a bridge between the two metal ions and attacks the carbonyl carbon of the substrate. The second molecule is a water molecule (Wat2) that is bound to the carboxyl group of Glu122 and functions as a proton donor in catalysis. The activity of the E122Q mutant was very low and it was only partially restored by the addition of ZnCl(2) or MnCl(2). In the E122Q mutant, k(cat) is drastically decreased, indicating that Glu122 is important for catalysis. X-ray crystallographic study and the atomic absorption spectrometry analysis of the E122Q mutant-substrate complex revealed that the drastic decrease of the activity of the E122Q was caused by not only the loss of one Zn ion at the Metal1 site but also a critical function of Glu122, which most likely exists for a proton transfer step through Wat2.


  • Organizational Affiliation

    Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Creatinine amidohydrolase
A, B, C, D, E
A, B, C, D, E, F
260Pseudomonas putidaMutation(s): 1 
EC: 3.5.2.10
UniProt
Find proteins for P83772 (Pseudomonas putida)
Explore P83772 
Go to UniProtKB:  P83772
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP83772
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 3 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
CRN
Query on CRN

Download Ideal Coordinates CCD File 
BA [auth F],
H [auth A],
M [auth B],
Q [auth C],
X [auth E]
N-[(E)-AMINO(IMINO)METHYL]-N-METHYLGLYCINE
C4 H9 N3 O2
CVSVTCORWBXHQV-UHFFFAOYSA-N
SO4
Query on SO4

Download Ideal Coordinates CCD File 
CA [auth F]
DA [auth F]
I [auth A]
J [auth A]
K [auth A]
CA [auth F],
DA [auth F],
I [auth A],
J [auth A],
K [auth A],
N [auth B],
O [auth B],
R [auth C],
S [auth C],
U [auth D],
V [auth D],
Y [auth E],
Z [auth E]
SULFATE ION
O4 S
QAOWNCQODCNURD-UHFFFAOYSA-L
ZN
Query on ZN

Download Ideal Coordinates CCD File 
AA [auth F]
G [auth A]
L [auth B]
P [auth C]
T [auth D]
AA [auth F],
G [auth A],
L [auth B],
P [auth C],
T [auth D],
W [auth E]
ZINC ION
Zn
PTFCDOFLOPIGGS-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.00 Å
  • R-Value Free: 0.212 
  • R-Value Work: 0.195 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 102.2α = 90
b = 152.7β = 90
c = 166.9γ = 90
Software Package:
Software NamePurpose
ADSCdata collection
CNSrefinement
HKL-2000data reduction
DPSdata reduction
CNSphasing

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2010-02-09
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Version format compliance
  • Version 1.2: 2014-01-22
    Changes: Database references
  • Version 1.3: 2021-11-10
    Changes: Database references, Derived calculations
  • Version 1.4: 2023-11-01
    Changes: Data collection, Refinement description