Theoretical Study of the Reaction Mechanism of Streptomyces coelicolor Type II Dehydroquinase

Abstract

The reaction mechanism of a type II dehydroquinase (DHQase) from Streptomyces coelicolor was investigated using molecular dynamics simulation and density functional theory (DFT) calculations. DHQase catalyzes the elimination of a water molecule from dehydroquinate (DHQ), a key step in the biosynthesis of aromatic amino acids in bacteria, fungi, and plants. In the DFT calculations, 10 models, containing up to 230 atoms, were used to investigate different proposals for the reaction mechanism, suggested on the basis of crystal structures and kinetic data. Probing the flexibility of the active site, molecular dynamics simulation reveals that deprotonated Tyr28 can act as the base that catalyzes the first reaction step, the proton abstraction of the pro-S proton at C2 of DHQ, and formation of the enolate intermediate. The computed barrier for the first transition state (TS1), 13-15 kcal/mol, is only slightly affected by the active site model used and is in good agreement with the corresponding experimental barrier of 13.4 kcal/mol for the rate-determining step. The previously proposed enol form of the intermediate is found to be significantly higher in energy than the enolate form and is thus thermodynamically not competitive. In the second and final reaction step, protonation of the hydroxyl group at C1 by His106 followed by water elimination, there is a substantial buildup of dipole moment due to the net transfer of a proton from His106 to Tyr28. A barrier for the second transition state (TS2) that fits well with the corresponding experimental barrier could only be found if the buildup of dipole moment is at least partly compensated during the second reaction step. We speculate that this could be facilitated by regeneration of the Tyr28 anion or by proton transfer to the vicinity of His106 before TS2 is reached. A revised mechanism for type II DHQase is discussed in light of the results of the present calculations.

Quantitative Comparison of Catalytic Mechanisms and Overall Reactions in Convergently Evolved Enzymes: Implications for Classification of Enzyme Function

“…Both pairs of enzymes had identical overall reactions, but only the former pair (beta-lactamases) had significant mechanistic similarity. It has been reported in the literature that type I and type II dehydroquinases catalyze the same chemical reaction but by completely different mechanisms [53],[56]. Our algorithm correctly aligns the C-O bond cleavage common to the sub-subclass, which is catalyzed in the sixth step of M0054 and in the second step of M0055 (Table S3).…”

Unraveling the kinetic diversity of microbial 3-dehydroquinate dehydratases of shikimate pathway

“…Alignments of type II DHQases revealed a number of conserved amino acid residues that are potentially important for catalytic efficiency and capacity. Specifically, the Tyr24 residue facilitated the proton abstraction from substrate, and then the His101 residues promoted the dehydrogenation by donating proton to 1-hydroxyl on C1 of substrate as a general acid (Blomberg et al 2009 ; Pan et al 2012 ). The residue His101was found in all the type II DHQases.…”

“…Many investigations of DHQases have been focused on their structures and catalytic mechanisms (Blomberg et al 2009 ; Bottomley et al 1996 ; Devi et al 2013 ; Lee et al 2002 ; Pan et al 2012 ; Roszak et al 2002 ), or on structure-based design of inhibitors to DHQase activity (Blanco et al 2012 ; 2014 ; Dias et al 2011 ; Peon et al 2010 ). These investigations have generated increasing numbers of DHQase structures with high resolution and have significantly advanced the understanding of DHQase catalytic mechanisms (Chaudhuri et al 1986 ; Deka et al 1994 ; Euverink et al 1992 ; Hawkins et al 1993 ; Lee et al 2003 ; Moore et al 1993 ; Roszak et al 2002 ; Singh and Christendat 2006 ).…”

“…These investigations have generated increasing numbers of DHQase structures with high resolution and have significantly advanced the understanding of DHQase catalytic mechanisms (Chaudhuri et al 1986 ; Deka et al 1994 ; Euverink et al 1992 ; Hawkins et al 1993 ; Lee et al 2003 ; Moore et al 1993 ; Roszak et al 2002 ; Singh and Christendat 2006 ). Type I DHQases catalyze the dehydrogenation of DHQ through a cis -elimination (Chaudhuri et al 1991 ; Gourley et al 1999 ; Leech et al 1995 ), while type II DHQases take a trans -elimination mechanism (Blomberg et al 2009 ; Bottomley et al 1996 ).…”

Inhibition of Shikimate Kinase and Type II Dehydroquinase for Antibiotic Discovery: Structure-Based Design and Simulation Studies

“…However, the suggested formation of an enol rather than an enolate intermediate did not explain the observed solvent isotope effects and proton inventory of Mt-DHQ2, in which there is a single proton contributing to this effect [41]. On the basis of results from quantum mechanical (QM) calculations using different Hamiltonians on Sc-DHQ2, Blomberg et al [43] subsequently suggested again the formation of enolate intermediate 26 because of its significantly lower energy compared to that of the enol intermediate.…”