Crystallization and preliminary crystallographic analysis of creatininase from Pseudomonas putida

Abstract

Creatininase (CrnA) from Pseudomonas putida is a homohexameric heat-stable enzyme composed of 259 amino acids per subunit. The molecular weight of each monomer is 28.4 kDa. The enzyme hydrolyses creatinine to yield creatine. Crystals of this protein have been grown from ethanol/PEG 8000. They belong to the monoclinic space group P2(1), with unit-cell parameters a = 74.8, b = 95.7, c = 116.9 A, alpha = gamma = 90, beta = 103.8 degrees. The diffraction limit is 2.5 A. The self-rotation function of the native data set is consistent with a CrnA hexamer in the asymmetric unit and suggests D(3) point-group symmetry of the enzyme.

Quantum Mechanics/Molecular Mechanics Simulations Identify the Ring-Opening Mechanism of Creatininase

“…The enzyme plays a key role in the bacterial degradation of creatinine 5 and participates in arginine and proline metabolism 6 . With growing industrial demand, considerable efforts have been made for decades to characterize the properties of creatininase from various microorganisms, including the bacterial enzyme from Pseudomonas putida 2,[7][8][9][10][11][12] . Since the first X-ray crystal structure of creatininase was reported in late 2002 by Beuth et al 9 , several structures have been characterized to understand the structure and function of the enzyme [9][10][11][12] .…”

“…The enzyme plays a key role in the bacterial degradation of creatinine and participates in arginine and proline metabolism . Because of growing industrial demand, considerable efforts have been made for decades to characterize the properties of creatininase from various microorganisms, including the bacterial enzyme from Pseudomonas putida . ,− Since the first X-ray crystal structure of creatininase was reported in late 2002 by Beuth et al, several structures have been characterized to understand the structure and function of the enzyme. − The structures showed that creatininase has a unique (βα) 4 structural fold compared to that of other members of the urease-related amidohydrolase superfamily, containing a binuclear metal center in each subunit. Usually, two zinc ions are located at the M1 and M2 sites (hereafter called Zn1 and Zn2, respectively) within the active site, ligated by five conserved amino acid residues (Glu34, His36, Asp45, His120, and Glu183) and two water molecules (see Scheme ).…”

“…In a Mn-activated enzyme (Mn–Zn enzyme), Mn1 has a square-pyramidal geometry bound to the three protein ligands, the substrate, and two water molecules (Wat1 and Wat2). Currently, a total of 13 crystal structures of the enzyme have been deposited in the Protein Data Bank (PDB), including the native enzyme , and the enzyme–inhibitor/product complex, , but none of them have a substrate bound. The lack of structural information about the Michaelis complex could lead to an incomplete understanding of the catalytic mechanism of creatininase.…”