Inactivation Mechanism of Glycerol Dehydration by Diol Dehydratase from Combined Quantum Mechanical/Molecular Mechanical Calculations

Abstract

Inactivation of diol dehydratase during the glycerol dehydration reaction is studied on the basis of quantum mechanical/molecular mechanical calculations. Glycerol is not a chiral compound but contains a prochiral carbon atom. Once it is bound to the active site, the enzyme adopts two binding conformations. One is predominantly responsible for the product-forming reaction (G(R) conformation), and the other primarily contributes to inactivation (G(S) conformation). Reactant radical is converted into a product and byproduct in the product-forming reaction and inactivation, respectively. The OH group migrates from C2 to C1 in the product-forming reaction, whereas the transfer of a hydrogen from the 3-OH group of glycerol to C1 takes place during the inactivation. The activation barrier of the hydrogen transfer does not depend on the substrate-binding conformation. On the other hand, the activation barrier of OH group migration is sensitive to conformation and is 4.5 kcal/mol lower in the G(R) conformation than in the G(S) conformation. In the OH group migration, Glu170 plays a critical role in stabilizing the reactant radical in the G(S) conformation. Moreover, the hydrogen bonding interaction between Ser301 and the 3-OH group of glycerol lowers the activation barrier in G(R)-TS2. As a result, the difference in energy between the hydrogen transfer and the OH group migration is reduced in the G(S) conformation, which shows that the inactivation is favored in the G(S) conformation.

Rational engineering of diol dehydratase enables 1,4-butanediol biosynthesis from xylose

“…High catalytic efficiency is exhibited toward 1,2-PD (a C3 diol), while limited catalytic efficiency is observed toward glycerol (a C3 triol) due to substrate inhibition (Yamanishi et al, 2012). An approach to address this inhibition has been developed from studying the protein-substrate complex, where it was inferred that the interaction of the C3 hydroxyl group of glycerol with S301 leads to inhibition of catalysis (Doitomi et al, 2012).…”

“…The K. oxytoca diol dehydratase has a complex coenzyme B12 dependent reaction mechanism, and is known to dehydrate its native substrate -1,2-propanediol (1,2-PD) efficiently and to undergo suicide inactivation in the presence of a C3 triol -glycerol (Doitomi et al, 2012). Since the catalytic mechanism of K. oxytoca diol dehydratase has been well studied with 1,2-PD and also with glycerol, we chose this enzyme as a candidate for rational engineering approaches to promote catalysis toward a non-native C4 triol -1,2,4-BTO.…”

Synthesis and Properties of Formylcobalamin and Propionylcobalamin, Novel Acylcobalamins

“…We recently discussed about the mechanism of inactivation of AdoCbl-dependent diol dehydratase by glycerol (18,19) and the possible involvement of formyl-Cbl in the inactivation process (20). Theoretical calculations suggest that glycerol inactivation of this enzyme begins with an intramolecular hydrogen transfer from the glycerol 3-OH group to C1 of the substrate radical, forming an O3-centered radical that decomposes to formaldehyde and a glycol radical (20). The formaldehyde produced might react with cob(II)alamin (Cbl II ) and the glycol radical, forming ethylene glycol and an inactive cobalamin like formyl-Cbl.…”

Glycerol as a Substrate and Inactivator of Coenzyme B12‐Dependent Diol Dehydratase

“…[15] Although these data point to possible inactivation by-products, none were isolated or characterized. [15] Using the crystal structure of B 12 -dDDH obtained with cyanocobalamin (CNCbl) instead of adenosylcobalamin (AdoCbl) as the cofactor, and GOL as the substrate (PDB code: 3AUJ [22] ), Doitomi et al [23] offered a suicide inactivation mechanism (Figure 1). According to the proposed mechanism, after the initial H-atom abstraction from C1 of GOL, inactivation occurs due to an intramolecular H-atom transfer from the hydroxyl group at C3 to the C1-centered radical, with concomitant formation of an O-centered radical at C3.…”

“…This large difference is at odds with the experimental findings that inactivation occurs~1 every 9000-19000 catalytic cycles. [14,15] Further, for the inactivation pathway proposed by Doitomi et al [23] to be operative, a hydrogen bond must form between the hydroxyl group at C3 of GOL (henceforth denoted as C3À OH) and Ser301 or Gln336. [22,23] The hydrogen bond results in formation of a five-membered ring in the transition state for the inactivation step.…”