Protein dynamics play a crucial role in various physiological functions, including enzyme catalysis. To explore conformational changes during enzyme catalysis, we conduct mix-and-inject serial crystallography, an advanced technique to capture time-resolved protein structures in real time, using the microcrystals of bacterial copper amine oxidase containing a protein-derived quinone cofactor. Within 50 ms of mixing the microcrystals (<4 μm) with a preferred substrate (2-phenylethylamine) under anaerobic conditions (reductive half-reaction), we observe domain movements associated with substrate binding and formation of a metastable reaction intermediate, a product Schiff-base of the quinone cofactor. At 100–1000 ms after mixing, conformational transition from aminoresorcinol to the semiquinone radical forms of the reduced cofactor progresses gradually, likely depending on the replacement of the product aldehyde by the next-cycle amine substrate that triggers the cofactor conformational change. Overall, this study provides structural insight into enzyme catalysis accompanying the active-site conformational changes that are hardly scrutinized by studies in solution. Protein motion is essential for biological processes like enzyme function. Here the authors report the time-resolved protein structures captured during catalysis of copper amine oxidase using mix-and-inject serial femtosecond X-ray crystallography.

Primary amines such as n-butylamine and 2-phenylethylamine serve as good nitrogen, carbon, and energy sources for bacterial growth. In many Gram-negative bacterial species, these amines are first oxidized by a periplasmic enzyme, quinohemoprotein amine dehydrogenase (QHNDH), encoded in an operon termed 'qhp', consisting of eight genes (qhpABCDEFGR). A gene predicted to encode an aldehyde dehydrogenase is also highly conserved in the vicinity of the qhp operon. In this study, we found that a 5'-upstream region of the aldehyde dehydrogenase gene in Paracoccus denitrificans has a high promoter activity that responds to n-butylamine supplementation in the culture medium, indicating co-regulation with the qhp genes by the transcriptional regulator QhpR. Hence, we designate this gene as the ninth member of the qhp operon, qhpH. Disruption of qhpH in P. denitrificans neither affected bacterial growth on primary amines, nor impaired QHNDH activity, suggesting the presence of another constitutive aldehyde dehydrogenase(s) compensating for the defect of qhpH. Nevertheless, heterologous expression of qhpH along with the eight qhp genes in an amine non-assimilating bacterium, Rhodobacter sphaeroides, significantly enhanced the growth on n-butylamine, as compared to the slow growth without qhpH. The recombinant QhpH purified from Escherichia coli cells showed high aldehyde dehydrogenase activities toward various aldehydes. These findings demonstrate that the qhpH gene encodes an aldehyde dehydrogenase with broad substrate specificity and is evolutionarily conserved with the qhp operon to play a role in an efficient metabolism of various primary amines in Gram-negative bacteria.

Determining precise locations of hydrogen atoms in enzyme active sites, especially those in the reaction intermediates, provides important information for understanding the structure-function relationships, such as a correlation between pH-dependency and protonation/deprotonation states of dissociable groups. To experimentally determine the coordinates of hydrogen atoms, we solved the neutron crystallographic structure of a catalytic intermediate of copper amine oxidase, containing a peptidyl quinone cofactor, topa quinone (TPQ), which is converted to a semiquinone radical form by anaerobic reaction with an amine substrate. Neutron diffraction data at 1.67 A resolution revealed the protonation/ deprotonation state of the active-site residues, including TPQ. The semiquinone form was doubly deprotonated at the 2-OH and 4OH positions. The surrounding hydrogen-bond network and the CH center dot center dot center dot p and NH center dot center dot center dot pi-like interactions with both sides of the TPQ ring were identified, affording a stabilization mechanism for the semiquinone radical structure. The pH-dependent conformational change of TPQ from an 'off-copper' aminoresorcinol to an 'on-copper' semiquinone was accompanied by protein/solvent proton exchange in the main-chain peptide bond of TPQ. Moreover, the neutron diffraction data disclosed the number of deuterium/ hydrogen atoms covalently attached to the terminal heavy atom of the ligand bound in a hydrophobic pocket adjacent to TPQ, which led to the conclusion that the product aldehyde was replaced by an amine substrate in the pocket. The amino group of the bound substrate interacted with the deprotonated side chain of a conserved aspartic acid residue that acts as a catalytic base. These findings demonstrate that the additional substrate binding triggers the conformational change of TPQ in the reductive half-reaction and makes the enzyme catalysis proceed into the subsequent oxidative half-reaction.

The mechanisms by which enzymes promote catalytic reactions efficiently through their structural changes remain to be fully elucidated. Recent progress in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers (XFELs) has made it possible to address these issues. In particular, mix-and-inject serial crystallography (MISC) is promising for the direct observation of structural changes associated with ongoing enzymic reactions. In this study, SFX measurements using a liquid-jet system were performed on microcrystals of bacterial copper amine oxidase anaerobically premixed with a substrate amine solution. The structure determined at 1.94 Å resolution indicated that the peptidyl quinone cofactor is in equilibrium between the aminoresorcinol and semiquinone radical intermediates, which accumulate only under anaerobic single-turnover conditions. These results show that anaerobic conditions were well maintained throughout the liquid-jet SFX measurements, preventing the catalytic intermediates from reacting with dioxygen. These results also provide a necessary framework for performing time-resolved MISC to study enzymic reaction mechanisms under anaerobic conditions.

Vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) in dorsal root ganglia (DRGs) are known to be upregulated and to contribute to the mechanisms of neuropathic pain following peripheral nerve injury. Moreover, transcription factor c-Jun regulates the expressions of both VIP and NPY in cultured DRG neurons. To elucidate the role of c-Jun in the induction of neuropathic pain hypersensitivity, we examined whether activated c-Jun affects pain behavior and the expressions of VIP and NPY following chronic constriction injury (CCI) of rat sciatic nerve. Intrathecal treatment with c-jun antisense oligodeoxynucleotides (AS-ODN) significantly reduced mechanical allodynia, but not thermal hyperalgesia following CCI. In addition, c-jun AS-ODN also suppressed the remarkable elevations of VIP and NPY mRNAs and the percentages of phosphorylated c-Jun-, VIP-, and NPY-immunoreactive neurons observed in DRGs following CCI. These results show that the activation of c-Jun in DRGs induces VIP and NPY upregulation and contributes to the pathogenesis of neuropathic pain following CCI.

Protein neutron crystallography is a powerful technique to determine the positions of H atoms, providing crucial biochemical information such as the protonation states of catalytic groups and the geometry of hydrogen bonds. Recently, the crystal structure of a bacterial copper amine oxidase was determined by joint refinement using X-ray and neutron diffraction data sets at resolutions of 1.14 and 1.72 Å, respectively [Murakawa et al. (2020). Proc. Natl Acad. Sci. USA, 117, 10818–10824]. While joint refinement is effective for the determination of the accurate positions of heavy atoms on the basis of the electron density, the structural information on light atoms (hydrogen and deuterium) derived from the neutron diffraction data might be affected by the X-ray data. To unravel the information included in the neutron diffraction data, the structure determination was conducted again using only the neutron diffraction data at 1.72 Å resolution and the results were compared with those obtained in the previous study. Most H and D atoms were identified at essentially the same positions in both the neutron-only and the X-ray/neutron joint refinements. Nevertheless, neutron-only refinement was found to be less effective than joint refinement in providing very accurate heavy-atom coordinates that lead to significant improvement of the neutron scattering length density map, especially for the active-site cofactor. Consequently, it was confirmed that X-ray/neutron joint refinement is crucial for determination of the real chemical structure of the catalytic site of the enzyme.

Bioconversion of peptidyl amino acids into enzyme cofactors is an important post-translational modification. Here, we report a flavoprotein, essential for biosynthesis of a protein-derived quinone cofactor, cysteine tryptophylquinone, contained in a widely distributed bacterial enzyme, quinohemoprotein amine dehydrogenase. The purified flavoprotein catalyzes the single-turnover dihydroxylation of the tryptophylquinone-precursor, tryptophan, in the protein substrate containing triple intra-peptidyl crosslinks that are pre-formed by a radical S-adenosylmethionine enzyme within the ternary complex of these proteins. Crystal structure of the peptidyl tryptophan dihydroxylase reveals a large pocket that may dock the protein substrate with the bound flavin adenine dinucleotide situated close to the precursor tryptophan. Based on the enzyme-protein substrate docking model, we propose a chemical reaction mechanism of peptidyl tryptophan dihydroxylation catalyzed by the flavoprotein monooxygenase. The diversity of the tryptophylquinone-generating systems suggests convergent evolution of the peptidyl tryptophan-derived cofactors in different proteins.

Quinohemoprotein amine dehydrogenase (QHNDH) containing a peptidyl quinone cofactor, cysteine tryptophylquinone, is produced in the periplasm of Gram-negative bacteria through an intricate process of post-translational modification that requires at least 8 genes including those encoding 3 nonidentical subunits and 3 modifying enzymes. Our heterologous expression study has revealed that the 8 genes are necessary and sufficient for the QHNDH biogenesis.

Recent advances in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers have paved the way for determining radiation-damage-free protein structures under nonfreezing conditions. However, the large-scale preparation of high-quality microcrystals of uniform size is a prerequisite for SFX, and this has been a barrier to its widespread application. Here, a convenient method for preparing high-quality microcrystals of a bacterial quinoprotein enzyme, copper amine oxidase from Arthrobacter globiformis, is reported. The method consists of the mechanical crushing of large crystals (5-15 mm3), seeding the crushed crystals into the enzyme solution and standing for 1 h at an ambient temperature of ∼26°C, leading to the rapid formation of microcrystals with a uniform size of 3-5 µm. The microcrystals diffracted X-rays to a resolution beyond 2.0 Å in SFX measurements at the SPring-8 Angstrom Compact Free Electron Laser facility. The damage-free structure determined at 2.2 Å resolution was essentially identical to that determined previously by cryogenic crystallography using synchrotron X-ray radiation.

Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper (Cu) amine oxidase (CAO), which contains a prosthetic Cu ion and a protein-derived redox cofactor, topa quinone (TPQ). We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a nonplanar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the Cu. Our findings show a refined active-site structure that gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions.

In the catalytic reaction of copper amine oxidase, the protein-derived redox cofactor topaquinone (TPQ) is reduced by an amine substrate to an aminoresorcinol form (TPQamr), which is in equilibrium with a semiquinone radical (TPQsq). The transition from TPQamr to TPQsq is an endothermic process, accompanied by a significant conformational change of the cofactor. We employed the humid air and glue-coating (HAG) method to capture the equilibrium mixture of TPQamr and TPQsq in noncryocooled crystals of the enzyme from Arthrobacter globiformis and found that the equilibrium shifts more toward TPQsq in crystals than in solution. Thermodynamic analyses of the temperature-dependent equilibrium also revealed that the transition to TPQsq is entropy-driven both in crystals and in solution, giving the thermodynamic parameters that led to experimental determination of the crystal packing effect. Furthermore, we demonstrate that the binding of product aldehyde to the hydrophobic pocket in the active site produces various equilibrium states among two forms of the product Schiff-base, TPQamr, and TPQsq, in a pH-dependent manner. The temperature-controlled HAG method provides a technique for thermodynamic analysis of conformational changes occurring in protein crystals that are hardly scrutinized by conventional cryogenic X-ray crystallography.