Divalent metal ion-based catalytic mechanism of the Nudix hydrolase Orf153 (YmfB) fromEscherichia coli

Abstract

YmfB from Escherichia coli is the Nudix hydrolase involved in the metabolism of thiamine pyrophosphate, an important compound in primary metabolism and a cofactor of many enzymes. In addition, it hydrolyzes (d)NTPs to (d)NMPs and inorganic orthophosphates in a stepwise manner. The structures of YmfB alone and in complex with three sulfates and two manganese ions determined by X-ray crystallography, when compared with the structures of other Nudix hydrolases such as MutT, Ap4Aase and DR1025, provide insight into the unique hydrolysis mechanism of YmfB. Mass-spectrometric analysis confirmed that water attacks the terminal phosphates of GTP and GDP sequentially. Kinetic analysis of binding-site mutants showed that no individual residue is absolutely required for catalytic activity, suggesting that protein residues do not participate in the deprotonation of the attacking water. Thermodynamic integration calculations show that a hydroxyl ion bound to two divalent metal ions attacks the phosphate directly without the help of a nearby catalytic base.

QM/MM study of the mechanism of reduction of 3-hydroxy-3-methylglutaryl coenzyme A catalyzed by human HMG-CoA reductase

“…The extremely anisotropic electrostatic environment, the limited number of well-known pK a s compared to the diversity of the environments, or the large number of titratable groups are some of the causes of the difficulty. Even though techniques such as thermodynamic integration could provide more reliable results for relative pK a s, 22 their accuracy (and that of the force fields) is still insufficient to result in solid conclusions about protonation states when subtle pK a differences are sought. Due to the lack of an accurate and reliable method for pK a prediction to resolve the controversy and since the differences in free energy between the different protonation states (for Glu559, His752 and Asp767) are smaller than the differences between the chemical mechanisms they led to (see Results and discussion), we opted to explore all possibilities and modeled explicitly three systems with different protonation states for Glu559, His752 and Asp767 residues instead of choosing a particular one without very solid calculations.…”

Metal ion coordination in the E. coli Nudix hydrolase dihydroneopterin triphosphate pyrophosphatase: New clues into catalytic mechanism

“…These residues are 3.8 or 3.5 Å from the metal-bridged solvent in those structures, respectively, similar to the 3.6 Å measured for Glu59 and the bridging solvent similarly positioned in DHNTPase. Alternative Glu residues have been proposed as the base in Ap4A orthologs [26, 29], but the case has also been made that a protein-derived general base is not necessary for divalent Nudix hydrolases given substantial pKa-lowering of the water by the metal ions in the cluster [34]. Indeed, on the basis of its level of sequence conservation and position in our structures in direct line with the apparent M2-M4-activated water and sulfur atom in the sulfate anion, deprotonation of the solvent in DHNTPase may not require a protein-based general base.…”

Rational design of a highly efficient catalytic system for the production of PAPS from ATP and its application in the synthesis of chondroitin sulfate

“…During the conversion of ATP to PAPS, two by‐products, PPi and ADP, inhibit the performance of ATPS and APSK (Bao et al, 2015). To eliminate such by‐product inhibition, PPA and Nudix hydrolase have been used as PPi and ADP hydrolases, respectively (Hong et al, 2014; W Xu et al, 2006). Previously, a complex ATP conversion system was constructed using 3‐bromopyruvic acid as a cheap substrate for PEP‐K + , which then acted as a phosphate donor for ADP (An et al, 2017).…”