Nonequivalence of Second Sphere "Noncatalytic" Residues in Pentaerythritol Tetranitrate Reductase in Relation to Local Dynamics Linked to H-Transfer in Reactions with NADH and NADPH Coenzymes.

Andreea I Iorgu,Jonathan P Waltho,Nigel S Scrutton,Colin Levy,Matthew J Cliff,Sam Hay,Nicola J Baxter

doi:10.1021/acscatal.8b02810

Andreea I Iorgu, Jonathan P Waltho + Show 5 more

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https://doi.org/10.1021/acscatal.8b02810

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Abstract

Many enzymes that catalyze hydride transfer reactions work via a mechanism dominated by quantum mechanical tunneling. The involvement of fast vibrational modes of the reactive complex is often inferred in these reactions, as in the case of the NAD(P)H-dependent pentaerythritol tetranitrate reductase (PETNR). Herein, we interrogated the H-transfer mechanism in PETNR by designing conservative (L25I and I107L) and side chain shortening (L25A and I107A) PETNR variants and using a combination of experimental approaches (stopped-flow rapid kinetics, X-ray crystallography, isotope/temperature dependence studies of H-transfer and NMR spectroscopy). X-ray data show subtle changes in the local environment of the targeted side chains but no major structural perturbation caused by mutagenesis of these two second sphere active site residues. However, temperature dependence studies of H-transfer revealed a coenzyme-specific and complex thermodynamic equilibrium between different reactive configurations in PETNR–coenzyme complexes. We find that mutagenesis of these second sphere “noncatalytic” residues affects differently the reactivity of PETNR with NADPH and NADH coenzymes. We attribute this to subtle, dynamic structural changes in the PETNR active site, the effects of which impact differently in the nonequivalent reactive geometries of PETNR−NADH and PETNR−NADPH complexes. This inference is confirmed through changes observed in the NMR chemical shift data for PETNR complexes with unreactive 1,4,5,6-tetrahydro-NAD(P) analogues. We show that H-transfer rates can (to some extent) be buffered through entropy–enthalpy compensation, but that use of integrated experimental tools reveals hidden complexities that implicate a role for dynamics in this relatively simple H-transfer reaction. Similar approaches are likely to be informative in other enzymes to understand the relative importance of (distal) hydrophobic side chains and dynamics in controlling the rates of enzymatic H-transfer.

Highlights

Understanding the physical basis of enzyme catalysis is important from a fundamental point of view and to drive applications in contemporary areas of research, such as biotechnology and synthetic biology
Direct coupling of fast dynamics to chemistry is mostly inferred from kinetic isotope effect (KIE) studies and supported by computational analysis such as transition path sampling.[20−22] The temperature dependence of KIEs is regarded as a “gold standard” for probing quantum mechanical tunneling (QMT) for H-transfer reactions and some interpretations have inferred a role for distance sampling dynamical contributions in facilitating the tunneling reaction[23−27]
The significant differences in chemical shift between pentaerythritol tetranitrate reductase (PETNR) and the PETNR−NAD(P)H4 complexes observed in the β-sheet containing A58 and the loop involving Y351, along with differences located at the top of the β-barrel that is in close proximity to flavin mononucleotide (FMN), suggest that NAD(P)H4 binding induces FMN repositioning within the active site for efficient H-transfer

Summary

■ INTRODUCTION

Understanding the physical basis of enzyme catalysis is important from a fundamental point of view and to drive applications in contemporary areas of research, such as biotechnology and synthetic biology. The first step (the reductive halfreaction) involves hydride transfer from the C4 pro-R hydrogen atom of the NAD(P)H coenzyme to the N5 atom of the noncovalently bound flavin mononucleotide (FMN) cofactor.[49] QMT contributes to this catalytic step,[43] and several experimental studies have suggested an involvement of fast (picosecond to nanosecond) dynamics in the H-transfer reaction.[23,50,51] Here, we interrogated the H-transfer mechanism in PETNR by designing four variants of two second sphere residues (L25 and I107) positioned along the FMN-NAD(P)H N5−C4 axis (Figure 1), as inferred from the structure of PETNR in complex with the unreactive NADH analogue 1,4,5,6-tetrahydro-NAD.[43] L25 is located below the FMN, with the side chain in van der Waals contact with the isoalloxazine ring of the FMN cofactor, while the side chain of I107 is positioned above the nicotinamide ring of the coenzyme These residues are “noncatalytic” and are assumed not to contribute to major active site electrostatic effects.

■ RESULTS AND DISCUSSION

■ CONCLUDING REMARKS

■ ACKNOWLEDGMENTS

■ REFERENCES