Crystal Structure of Creatininase from Pseudomonas putida: A Novel Fold and a Case of Convergent Evolution

Crystal Structures of Creatininase Reveal the Substrate Binding Site and Provide an Insight into the Catalytic Mechanism

“…In our version of the reaction mechanism, the water-adding step is basically consistent with the mechanism proposed by Beuth et al, 15 but the ring-opening step is quite different from that proposed by Beuth et al Their reaction mechanism was proposed based on computer modeling and structural comparisons with other amidohydrolases, rather than the structural evidence of enzyme -substrate/inhibitor complexes. Consequently, their reaction mechanism is similar to that of other amidohydrolases.…”

“…Based on the high-resolution crystal structure of the creatininase -creatine complex, we propose a new two-step catalytic mechanism possibly common to creatininases in which a water molecule (Wat1), possibly hydroxide, acts as the attacking nucleophile in the water-adding step and another water molecule (Wat2) acts as the catalytic acid in the ring-opening step. In our reaction mechanism, the mechanism of the water-adding step is basically consistent with the mechanism proposed by Beuth et al, 15 but the mechanism of the ringopening step is quite different from that proposed by Beuth et al…”

“…14 Just before the submission of this paper for publication, Beuth et al reported the crystal structure of creatininase from P. putida, which had 97% amino acid sequence identity with our enzyme. 15 In the structure reported by Beuth et al, however, creatininase was crystallized without a bound substrate or inhibitor. Although Beuth and colleagues did propose a two-step reaction mechanism of creatininase in which metal-bridged hydroxide acts as the attacking nucleophile in the wateradding step and the His178 acts as the catalytic acid in the ring-opening step, based on the computer modeling of the reaction intermediate, there was a problem in the mechanism of the second step, which was that the side-chain of His178 was too far from the substrate molecule.…”

“…Although Beuth and colleagues did propose a two-step reaction mechanism of creatininase in which metal-bridged hydroxide acts as the attacking nucleophile in the wateradding step and the His178 acts as the catalytic acid in the ring-opening step, based on the computer modeling of the reaction intermediate, there was a problem in the mechanism of the second step, which was that the side-chain of His178 was too far from the substrate molecule. 15 This problem must be an artifact resulting from the coarse modeling procedure, because Beuth et al did not have the crystal structure of creatininase complexed with a bound substrate or inhibitors. Thus, understanding the three-dimensional structure of creatininase complexed with a bound substrate or inhibitor is essential for clarifying the substrate binding mode and the catalytic mechanism of the enzyme.…”

Substitution of Glu122 by Glutamine Revealed the Function of the Second Water Molecule as a Proton Donor in the Binuclear Metal Enzyme Creatininase

“…The threedimensional structures of these enzymes have been reported. [8][9][10][11][12][13][14][15] We have determined the structures of the wildtype and Mn-activated enzymes. Two metal ions have been found at an active site in each subunit; two zinc ions have been found in the wild-type enzyme.…”