Re-Face Stereospecificity of Methylenetetrahydromethanopterin and Methylenetetrahydrofolate Dehydrogenases is Predetermined by Intrinsic Properties of the Substrate

Abstract

Four different dehydrogenases are known that catalyse the reversible dehydrogenation of N5,N10-methylenetetrahydromethanopterin (methylene-H4MPT) or N5,N10-methylenetetrahydrofolate (methylene-H4F) to the respective N5,N10-methenyl compounds. Sequence comparison indicates that the four enzymes are phylogenetically unrelated. They all catalyse the Re-face-stereospecific removal of the pro-R hydrogen atom of the coenzyme's methylene group. The Re-face stereospecificity is in contrast to the finding that in solution the pro-S hydrogen atom of methylene-H4MPT and of methylene-H4F is more reactive to heterolytic cleavage. For a better understanding we determined the conformations of methylene-H4MPT in solution and when enzyme-bound by using NMR spectroscopy and semiempirical quantum mechanical calculations. For the conformation free in solution we find an envelope conformation for the imidazolidine ring, with the flap at N10. The methylene pro-S C-H bond is anticlinal and the methylene pro-R C-H bond is synclinal to the lone electron pair of N10. Semiempirical quantum mechanical calculations of heats of formation of methylene-H4MPT and methylene-H4F indicate that changing this conformation into an activated one in which the pro-S C-H bond is antiperiplanar, resulting in the preformation of the leaving hydride, would require a deltadeltaH(f) of +53 kJ mol-1 for methylene-H4MPT and of +51 kJ mol-1 for methylene-H4F. This is almost twice the energy required to force the imidazolidine ring in the enzyme-bound conformation of methylene-H4MPT (+29 kJ mol-1) or of methylene-H4F (+35 kJ mol-1) into an activated conformation in which the pro-R hydrogen atom is antiperiplanar to the lone electron pair of N10. The much lower energy for pro-R hydrogen activation thus probably predetermines the Re-face stereospecificity of the four dehydrogenases. Results are also presented explaining why the chemical reduction of methenyl-H4MPT+ and methenyl-H4F+ with NaBD4 proceeds Si-face-specific, in contrast to the enzyme-catalysed reaction.

The Crystal Structure of an [Fe]‐Hydrogenase–Substrate Complex Reveals the Framework for H2 Activation

“…The head part is in a relatively “extended” conformation and fit into the active site cleft (Supporting Information, Figure S1B). This conformation is definitely different from the “bent” NMR spectroscopy structure in solution (Supporting Information, Figure S3) 18. In contrast, the overall conformation of the head part resembles that bound to [Fe]‐hydrogenase from Methanothermobacter marburgensis previously determined by NMR spectroscopy18, 19 although a closer look reveals functionally relevant differences, in particular, at N5 and N10 of the imidazolidine ring.…”

The Crystal Structure of the Apoenzyme of the Iron–Sulphur Cluster-free Hydrogenase

“…[8][9][10] In the reaction, a hydride ion is transferred stereospecifically from H 2 to C 14a of methenyl-H 4 MPT C , which has carbocationic character. [11][12][13] The enzyme exhibits a ternary complex catalytic mechanism. It does not catalyse the reduction of viologen dyes with H 2 or, in the absence of methenyl-H 4 MPT C , the exchange of H 2 with protons of water, nor does the inter-conversion of para H 2 and ortho H 2 occur at significant rates, 2,14,15 which are reactions actively catalysed by [NiFe] and [FeFe]-hydrogenases.…”

Drug Design for G‐Protein‐Coupled Receptors by a Ligand‐Based NMR Method

“…Having demonstrated the specificity and dose dependency of the binding events, we set out to investigate whether we could derive data on the bioactive conformation and the relative orientation of two ligands in the binding site of GPR40 using transfer‐NOE and interligand NOE methods (INPHARMA) 3. 4, 11 Ligands B and C were chosen for these studies as both had been chosen previously for chemical lead optimization.…”