Judith P. Klinman

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

Enzyme dynamics and hydrogen tunnelling in a thermophilic alcohol dehydrogenase

Biological catalysts (enzymes) speed up reactions by many orders of magnitude using fundamental physical processes to increase chemical reactivity. Hydrogen tunnelling has increasingly been found to contribute to enzyme reactions at room temperature. Tunnelling is the phenomenon by which a particle transfers through a reaction barrier as a result of its wave-like property. In reactions involving small molecules, the relative importance of tunnelling increases as the temperature is reduced. We have now investigated whether hydrogen tunnelling occurs at elevated temperatures in a biological system that functions physiologically under such conditions. Using a thermophilic alcohol dehydrogenase (ADH), we find that hydrogen tunnelling makes a significant contribution at 65 °C; this is analogous to previous findings with mesophilic ADH at 25 °C ( ref. 5). Contrary to predictions for tunnelling through a rigid barrier, the tunnelling with the thermophilic ADH decreases at and below room temperature. These findings provide experimental evidence for a role of thermally excited enzyme fluctuations in modulating enzyme-catalysed bond cleavage.

A Crosslinked Cofactor in Lysyl Oxidase: Redox Function for Amino Acid Side Chains

A previously unknown redox cofactor has been identified in the active site of lysyl oxidase from the bovine aorta. Edman sequencing, mass spectrometry, ultraviolet-visible spectra, and resonance Raman studies showed that this cofactor is a quinone. Its structure is derived from the crosslinking of the ϵ-amino group of a peptidyl lysine with the modified side chain of a tyrosyl residue, and it has been designated lysine tyrosylquinone. This quinone appears to be the only example of a mammalian cofactor formed from the crosslinking of two amino acid side chains. This discovery expands the range of known quino-cofactor structures and has implications for the mechanism of their biogenesis.

The Copper-Enzyme Family of Dopamine β-Monooxygenase and Peptidylglycine α-Hydroxylating Monooxygenase: Resolving the Chemical Pathway for Substrate Hydroxylation

Dopamine -monooxygenase (D M)2 and peptidylglycine -hydroxylating monooxygenase (PHM) belong to a small class of copper proteins found exclusively in higher eukaryotes. These physiologically important enzymes catalyze the transformation of dopamine to norepinephrine (D M) (Equation 1) and C-terminal glycine-extended peptides to their -hydroxylated products (PHM) (Equation 2). Although their substrate specificities are grossly different,

Liver alcohol dehydrogenase.

The article deals with the structure and function of liver alcohol dehydrogenase and reviews mainly literature published after 1979, i.e., summarizes progress made in the field since Klinman presented her review on alcohol dehydrogenases. The emphasis will be on high-resolution crystallographic data, results obtained with metal-substituted enzyme derivatives, and on the mechanism and pH dependence of the catalytic reaction.

Hydrogen tunneling in biology

The mechanistic details of hydrogen transfer in biological systems are not fully understood. The traditional approach has been to use semiclassical transition-state theory. This theory cannot explain many experimental findings, however, so different approaches that emphasize the importance of quantum mechanics and dynamic effects should also be considered.

Hydrogen tunneling links protein dynamics to enzyme catalysis.

The relationship between protein dynamics and function is a subject of considerable contemporary interest. Although protein motions are frequently observed during ligand binding and release steps, the contribution of protein motions to the catalysis of bond making/breaking processes is more difficult to probe and verify. Here, we show how the quantum mechanical hydrogen tunneling associated with enzymatic C-H bond cleavage provides a unique window into the necessity of protein dynamics for achieving optimal catalysis. Experimental findings support a hierarchy of thermodynamically equilibrated motions that control the H-donor and -acceptor distance and active-site electrostatics, creating an ensemble of conformations suitable for H-tunneling. A possible extension of this view to methyl transfer and other catalyzed reactions is also presented. The impact of understanding these dynamics on the conceptual framework for enzyme activity, inhibitor/drug design, and biomimetic catalyst design is likely to be substantial.

Catalysis of electron transfer during activation of O2 by the flavoprotein glucose oxidase.

Two prototropic forms of glucose oxidase undergo aerobic oxidation reactions that convert FADH− to FAD and form H2O2 as a product. Limiting rate constants of kcat/KM(O2) = (5.7 ± 1.8) × 102 M−1⋅s−1 and kcat/KM(O2) = (1.5 ± 0.3) × 106 M−1⋅s−1 are observed at high and low pH, respectively. Reactions exhibit oxygen-18 kinetic isotope effects but no solvent kinetic isotope effects, consistent with mechanisms of rate-limiting electron transfer from flavin to O2. Site-directed mutagenesis studies reveal that the pH dependence of the rates is caused by protonation of a highly conserved histidine in the active site. Temperature studies (283–323 K) indicate that protonation of His-516 results in a reduction of the activation energy barrier by 6.0 kcal⋅mol−1 (0.26 eV). Within the context of Marcus theory, catalysis of electron transfer is attributed to a 19-kcal⋅mol−1 (0.82 eV) decrease in the reorganization energy and a much smaller 2.2-kcal⋅mol−1 (0.095 eV) enhancement of the reaction driving force. An explanation is advanced that is based on changes in outer-sphere reorganization as a function of pH. The active site is optimized at low pH, but not at high pH or in the H516A mutant where rates resemble the uncatalyzed reaction in solution.

Evidence That Dioxygen and Substrate Activation Are Tightly Coupled in Dopamine β-Monooxygenase IMPLICATIONS FOR THE REACTIVE OXYGEN SPECIES

Oxygen activation occurs at a wide variety of enzyme active sites. Mechanisms previously proposed for the copper monooxygenase, dopamine β-monooxygenase (DβM), involve the accumulation of an activated oxygen intermediate with the properties of a copper-peroxo or copper-oxo species before substrate activation. These are reminiscent of the mechanism of cytochrome P-450, where a heme iron stabilizes the activated O2 species. Herein, we report two experimental probes of the activated oxygen species in DβM. First, we have synthesized the substrate analog, β,β-difluorophenethylamine, and examined its capacity to induce reoxidation of the prereduced copper sites of DβM upon mixing with O2 under rapid freeze-quench conditions. This experiment fails to give rise to an EPR-detectable copper species, in contrast to a substrate with a C–H active bond. This indicates either that the reoxidation of the enzyme-bound copper sites in the presence of O2 is tightly linked to C-H activation or that a diamagnetic species \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Cu(II)}-\mathrm{O}_{2}^{{\cdot}}\) \end{document} has been formed. In the context of the open and fully solvent-accessible active site for the homologous peptidylglycine-α-hydroxylating monooxygenase and by analogy to cytochrome P-450, the accumulation of a reduced and activated oxygen species in DβM before C-H cleavage would be expected to give some uncoupling of oxygen and substrate consumption. We have, therefore, examined the degree to which O2 and substrate consumption are coupled in DβM using both end point and initial rate experimental protocols. With substrates that differ by more than three orders of magnitude in rate, we fail to detect any uncoupling of O2 uptake from product formation. We conclude that there is no accumulation of an activated form of O2 before C-H abstraction in the DβM and peptidylglycine-α-hydroxylating monooxygenase class of copper monooxygenases, presenting a mechanism in which a diamagnetic Cu(II)-superoxo complex, formed initially at very low levels, abstracts a hydrogen atom from substrate to generate Cu(II)-hydroperoxo and substrate-free radical as intermediates. Subsequent participation of the second copper site per subunit completes the reaction cycle, generating hydroxylated product and water.

Copper amine oxidase from Hansenula polymorpha: the crystal structure determined at 2.4 å resolution reveals the active conformation

BACKGROUND Copper-containing amine oxidases (CAOs) are widespread in nature. These enzymes oxidize primary amine substrates to the aldehyde product, reducing molecular oxygen to hydrogen peroxide in the process. CAOs contain one type 2 copper atom and topaquinone (TPQ), a modified tyrosine sidechain utilized as a redox cofactor. The methylamine oxidase from the yeast Hansenula polymorpha (HPAO) is an isoform of CAO with a preference for small aliphatic amine or phenethylamine substrates. The enzyme is dimeric with a subunit molecular weight of 78 kDa. Structural studies are directed at understanding the basis for cofactor biogenesis and catalytic efficiency. RESULTS The X-ray crystal structure of HPAO has been solved at 2.4 A resolution by a combination of molecular replacement and single isomorphous replacement followed by refinement using sixfold symmetry averaging. The electron density at the catalytic site shows that the TPQ conformation corresponds to that of the active form of the enzyme. Two channels, one on either side of TPQ, are observed in the structure that provide access between the active site and the bulk solvent. CONCLUSIONS The structure shows TPQ in a position poised for catalysis. This is the first active CAO structure to reveal this conformation and may help further our understanding of the catalytic mechanism. On the substrate side of TPQ a water-containing channel leading to the protein surface can serve as an entrance or exit for substrate and product. On the opposite side of TPQ there is direct access from the bulk solvent of the dimer interface by which molecular oxygen may enter and hydrogen peroxide depart. In addition, a network of conserved water molecules has been identified which may function in the catalytic mechanism.