Cryptic enzymatic assembly of peptides armed with ß-lactone warheads.

Nature has evolved biosynthetic pathways to molecules possessing reactive warheads that inspired the development of many therapeutic agents, including penicillin antibiotics. Peptides armed with electrophilic warheads have proven to be particularly effective covalent inhibitors, providing essential antimicrobial, antiviral and anticancer agents. Here we provide a full characterization of the pathways that nature deploys to assemble peptides with ß-lactone warheads, which are potent proteasome inhibitors with promising anticancer activity. Warhead assembly involves a three-step cryptic methylation sequence, which is likely required to reduce unfavorable electrostatic interactions during the sterically demanding ß-lactonization. Amide-bond synthetase and adenosine triphosphate (ATP)-grasp enzymes couple amino acids to the ß-lactone warhead, generating the bioactive peptide products. After reconstituting the entire pathway to ß-lactone peptides in vitro, we go on to deliver a diverse range of analogs through enzymatic cascade reactions. Our approach is more efficient and cleaner than the synthetic methods currently used to produce clinically important warhead-containing peptides.

Photoinduced Electron Transfer from a 1,4,5,6-Tetrahydro Nicotinamide Adenine Dinucleotide (Phosphate) Analogue to Oxidized Flavin in an Ene-Reductase Flavoenzyme.

Recent reports have described the use of ene-reductase flavoenzymes to catalyze non-natural photochemical reactions. These studies have focused on using reduced flavoenzyme, yet oxidized flavins have superior light harvesting properties. In a binary complex of the oxidized ene-reductase pentaerythritol tetranitrate reductase with the nonreactive nicotinamide coenzyme analogs 1,4,5,6-tetrahydro NAD(P)H, visible photoexcitation of the flavin mononucleotide (FMN) leads to one-electron transfer from the NAD(P)H4 to FMN, generating a NAD(P)H4 cation radical and anionic FMN semiquinone. This electron transfer occurs in ∼1 ps and appears to kinetically outcompete reductive quenching from aromatic residues in the active site. Time-resolved infrared measurements show that relaxation processes appear to be largely localized on the FMN and the charge-separated state is short-lived, with relaxation, presumably via back electron transfer, occurring over ∼3-30 ps. While this demonstrates the potential for non-natural photoactivity, useful photocatalysis will likely require longer-lived excited states, which may be accessible by enzyme engineering and/or a judicious choice of substrate.

Solution-State Inter-Copper Distribution of Redox Partner-Linked Copper Nitrite Reductases: A Pulsed Electron-Electron Double Resonance Spectroscopy Study.

Copper nitrite reductases (CuNiRs) catalyze the reduction of nitrite to form nitric oxide. In recent years, new classes of redox partner linked CuNiRs have been isolated and characterized by crystallographic techniques. Solution-state biophysical studies have shed light on the complex catalytic mechanisms of these enzymes and implied that protein dynamics may play a role in CuNiR catalysis. To investigate the structural, dynamical, and functional relationship of these CuNiRs, we have used protein reverse engineering and pulsed electron-electron double resonance (PELDOR) spectroscopy to determine their solution-state inter-copper distributions. Data show the multidimensional conformational landscape of this family of enzymes and the role of tethering in catalysis. The importance of combining high-resolution crystallographic techniques and low-resolution solution-state approaches in determining the structures and mechanisms of metalloenzymes is emphasized by our approach.

Ultrafast Vibrational Energy Transfer between Protein and Cofactor in a Flavoenzyme.

Protein motions and enzyme catalysis are often linked. It is hypothesized that ultrafast vibrations (femtosecond-picosecond) enhance the rate of hydride transfer catalyzed by members of the old yellow enzyme (OYE) family of ene-reductases. Here, we use time-resolved infrared (TRIR) spectroscopy in combination with stable "heavy" isotopic labeling (2H, 13C, 15N) of protein and/or cofactor to probe the vibrational energy transfer (VET) between pentaerythritol tetranitrate reductase (a member of the OYE family) and its noncovalently bound flavin mononucleotide (FMN) cofactor. We show that when the FMN cofactor is photoexcited with visible light, vibrational energy is transferred from the flavin to the surrounding protein environment on the picosecond timescale. This finding expands the scope of VET investigation in proteins, which are limited by suitable intrinsic probes, and may have implications in the understanding of the mechanism of recently discovered photoactive flavoenzymes.

What are the signatures of tunnelling in enzyme-catalysed reactions?

While it is well established that thermally-activated quantum mechanical tunnelling of light particles (electrons and light atoms, typically hydrogen) plays a role in many enzyme-catalysed reactions, there are few definitive experimental signatures of atomic tunnelling and no clear methods of directly estimating the relative tunnelling contribution from typical experimental data. As most enzyme reactions involve the binding/capture of freely diffusing substrate(s), reactions are typically initiated by mixing and experimental conditions must then be compatible with liquid water (the solvent). This precludes the classic test of tunnelling: the observation of temperature-independent rate constants at cryogenic temperatures. Instead, H-tunnelling is usually inferred from kinetic isotope effects that are larger than the semiclassical limit. Often, the temperature dependence of the reaction is also measured over the experimentally accessible range (∼278-313 K for mesophilic enzymes), with resulting data analysed and interpreted using variations of Arrhenius, Eyring or Marcus theory. The apparent Arrhenius and Eyring activation parameters allow some quantitative comparison of different reactions, but do not directly provide any information about tunnelling, while the validity of parameters derived from non-adiabatic models such as Marcus theory are questionable due to the partially adiabatic nature of these reactions. Here, we use the correlation found between apparent activation enthalpy and entropy across several series of enzyme variants and tunnelling contributions determined using computational chemistry in an attempt to question and define new signatures of hydrogen tunnelling, which can be used to interpret typical experimental kinetic data measured for enzyme-catalysed reactions.

Isotopically labeled flavoenzymes and their uses in probing reaction mechanisms.

The incorporation of stable isotopes into proteins is beneficial or essential for a range of experiments, including NMR, neutron scattering and reflectometry, proteomic mass spectrometry, vibrational spectroscopy and "heavy" enzyme kinetic isotope effect (KIE) measurements. Here, we present detailed protocols for the stable isotopic labeling of pentaerythritol tetranitrate reductase (PETNR) via recombinant expression in E. coli. PETNR is an ene-reductase belonging to the Old Yellow Enzyme (OYE) family of flavoenzymes, and is regarded as a model system for studying hydride transfer reactions. Included is a discussion of how efficient back-exchange of amide protons in the protein core can be achieved and how the intrinsic flavin mononucleotide (FMN) cofactor can be exchanged, allowing the production of isotopologues with differentially labeled protein and cofactor. In addition to a thorough description of labeling strategies, we briefly exemplify how data analysis and interpretation of "heavy" enzyme KIEs can be performed.

Solvent-slaved protein motions accompany proton coupled electron transfer reactions catalysed by copper nitrite reductase.

Through the use of time-resolved pH-jump spectroscopy, we demonstrate how proton transfer is coupled to inter-copper electron transfer in a copper nitrite reductase (CuNiR). Combined use of electron paramagnetic resonance spectroscopy with solvent viscosity- and pressure-dependence pH-jump stopped-flow spectroscopy is used to show that solvent-slaved protein motions are linked to this proton coupled electron transfer step in CuNiR.

Selectivity through discriminatory induced fit enables switching of NAD(P)H coenzyme specificity in Old Yellow Enzyme ene-reductases.

Most ene-reductases belong to the Old Yellow Enzyme (OYE) family of flavin-dependent oxidoreductases. OYEs use nicotinamide coenzymes as hydride donors to catalyze the reduction of alkenes that contain an electron-withdrawing group. There have been many investigations of the structures and catalytic mechanisms of OYEs. However, the origin of coenzyme specificity in the OYE family is unknown. Structural NMR and X-ray crystallographic data were used to rationally design variants of two OYEs, pentaerythritol tetranitrate reductase (PETNR) and morphinone reductase (MR), to discover the basis of coenzyme selectivity. PETNR has dual-specificity and reacts with NADH and NADPH; MR accepts only NADH as hydride donor. Variants of a ß-hairpin motif in an active site loop of both these enzymes were studied using stopped-flow spectroscopy. Specific attention was placed on the potential role of arginine residues within the ß-hairpin motif. Mutagenesis demonstrated that Arg130 governs the preference of PETNR for NADPH, and that Arg142 interacts with the coenzyme pyrophosphate group. These observations were used to switch coenzyme specificity in MR by replacing either Glu134 or Leu146 with arginine residues. These variants had increased (~15-fold) affinity for NADH. Mutagenesis enabled MR to accept NADPH as a hydride donor, with E134R MR showing a significant (55-fold) increase in efficiency in the reductive half-reaction, when compared to the essentially unreactive wild-type enzyme. Insight into the question of coenzyme selectivity in OYEs has therefore been addressed through rational redesign. This should enable coenzyme selectivity to be improved and switched in other OYEs.

Unexpected Roles of a Tether Harboring a Tyrosine Gatekeeper Residue in Modular Nitrite Reductase Catalysis.

It is generally assumed that tethering enhances rates of electron harvesting and delivery to active sites in multidomain enzymes by proximity and sampling mechanisms. Here, we explore this idea in a tethered 3-domain, trimeric copper-containing nitrite reductase. By reverse engineering, we find that tethering does not enhance the rate of electron delivery from its pendant cytochrome c to the catalytic copper-containing core. Using a linker that harbors a gatekeeper tyrosine in a nitrite access channel, the tethered haem domain enables catalysis by other mechanisms. Tethering communicates the redox state of the haem to the distant T2Cu center that helps initiate substrate binding for catalysis. It also tunes copper reduction potentials, suppresses reductive enzyme inactivation, enhances enzyme affinity for substrate, and promotes intercopper electron transfer. Tethering has multiple unanticipated beneficial roles, the combination of which fine-tunes function beyond simplistic mechanisms expected from proximity and restrictive sampling models.

1H, 15N and 13C backbone resonance assignments of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2.

Pentaerythritol tetranitrate reductase (PETNR) is a flavoenzyme possessing a broad substrate specificity and is a member of the Old Yellow Enzyme family of oxidoreductases. As well as having high potential as an industrial biocatalyst, PETNR is an excellent model system for studying hydrogen transfer reactions. Mechanistic studies performed with PETNR using stopped-flow methods have shown that tunneling contributes towards hydride transfer from the NAD(P)H coenzyme to the flavin mononucleotide (FMN) cofactor and fast protein dynamics have been inferred to facilitate this catalytic step. Herein, we report the near-complete 1H, 15N and 13C backbone resonance assignments of PETNR in a stoichiometric complex with the FMN cofactor in its native oxidized form, which were obtained using heteronuclear multidimensional NMR spectroscopy. A total of 97% of all backbone resonances were assigned, with 333 out of a possible 344 residues assigned in the 1H-15N TROSY spectrum. This is the first report of an NMR structural study of a flavoenzyme from the Old Yellow Enzyme family and it lays the foundation for future investigations of functional dynamics in hydride transfer catalytic mechanism.

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.

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.