Catalytic Roles of the Metal Ion in the Substrate-Binding Site of Coenzyme B₁₂-Dependent Diol Dehydratase

Abstract

Functions of the metal ion in the substrate-binding site of diol dehydratase are studied on the basis of quantum mechanical/molecular mechanical (QM/MM) calculations. The metal ion directly coordinates to substrate and is essential for structural retention and substrate binding. The metal ion has been originally assigned to the K(+) ion; however, QM/MM computations indicate that Ca(2+) ion is more reasonable as the metal ion because calculated Ca-O distances better fit to the coordination distances in X-ray crystal structures rather than calculated K-O distances. The activation energy for the OH group migration, which is essential in the conversion of diols to corresponding aldehydes, is sensitive to the identity of the metal ion. For example, the spectator OH group of substrate is fully deprotonated by Glu170 in the transition state for the OH group migration in the Ca-contained QM/MM model, and therefore the barrier height is significantly decreased in the model having Ca(2+) ion. On the other hand, the deprotonation of the spectator OH group cannot effectively be triggered by the K(+) ion. Moreover, in the hydrogen recombination, the most energy-demanding step is more favorable in the Ca-contained model. The proposal that the Ca(2+) ion should be involved in the substrate-binding site is consistent with an observed large deuterium kinetic isotope effect of 10, which indicates that C-H bond activation is involved in the rate-determining step. Asp335 is found to have a strong anticatalytic effect on the OH group migration despite its important role in substrate binding. The synergistic interplay of the O-C bond cleavage by Ca(2+) ion and the deprotonation of the spectator OH group by Glu170 is required to overcome the anticatalytic effect of Asp335.

Radical SAM enzyme QueE defines a new minimal core fold and metal-dependent mechanism

“…These structures suggest that Na + and Mn 2+ cannot substitute for Mg 2+ due to their differential interactions with substrate, either the loss of coordination with the carbonyl moiety of substrate or the gain of interaction with a ring nitrogen, respectively. Divalent and monovalent metals are known to assist in adenosylcobalamindependent chemistry 33–35 , but the QueE reaction is the first characterized example for the AdoMet radical superfamily. Importantly, no sequence motif exists for the Mg 2+ binding site with only one protein residue involved in coordination.…”

Identification and Characterization ofRe-Citrate Synthase in Syntrophus aciditrophicus

“…S2 in the supplemental material) tidine residue performs these tasks (41). Recently, Ca 2ϩ was assigned as the metal ion instead of the K ϩ ion in coenzyme B 12 -dependent diol dehydratase on the basis of quantum mechanical/ molecular mechanical calculations (42) and by removal with EDTA followed by reconstitution (43). Even though Ca 2ϩ ion was detected in Re-citrate synthase by ICP-OES, we still do not have a clear picture of its coordination in the enzyme and catalytic function.…”

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

“…The metal ion plays an essential role in catalysis by (a) holding the substrate in the favorable orientation leading to initiation of catalysis, (b) allowing hydrogen abstraction from the substrate and OH group migration (Kamachi et al, 2011). It was determined that the distance between the metal ion and O1 of 1,2-PD was 2.5 Å, representative of an optimal distance for catalysis to proceed (Kamachi et al, 2011).…”

“…The metal ion plays an essential role in catalysis by (a) holding the substrate in the favorable orientation leading to initiation of catalysis, (b) allowing hydrogen abstraction from the substrate and OH group migration (Kamachi et al, 2011). It was determined that the distance between the metal ion and O1 of 1,2-PD was 2.5 Å, representative of an optimal distance for catalysis to proceed (Kamachi et al, 2011). From these studies we inferred that a distance close to 2.5 Å between the metal ion in the active site and O1 of the substrate is critical to catalysis, as an increased distance may not be thermodynamically favorable to catalysis (hydrogen abstraction, hydrogen back-abstraction or OH group migration) leading to possible substrate inhibition.…”

“…We developed an in silico screening approach to (a) identify prospective targets within the 5 Å radius that would influence substrate orientation, (b) serve as a screening platform to study the influence of substitutions on substrate (1,2,4-BTO) orientation and distance from metal ion. Residues directly involved in catalysis were first eliminated as candidates (Q141, E170, E221, H143, Q296, D335 and S362) (Kamachi et al, 2011). T222, V300 and F374, were identified as potential targets initially, as they are within the 5 Å radius, and not involved in catalysis directly.…”