Optobiochemistry: Genetically Encoded Control of Protein Activity by Light.

Optobiochemical control of protein activities allows the investigation of protein functions in living cells with high spatiotemporal resolution. Over the last two decades, numerous natural photosensory domains have been characterized and synthetic domains engineered and assembled into photoregulatory systems to control protein function with light. Here, we review the field of optobiochemistry, categorizing photosensory domains by chromophore, describing photoregulatory systems by mechanism of action, and discussing protein classes frequently investigated using optical methods. We also present examples of how spatial or temporal control of proteins in living cells has provided new insights not possible with traditional biochemical or cell biological techniques.

Structure of human phagocyte NADPH oxidase in the activated state.

Phagocyte NADPH oxidase, a protein complex with a core made up of NOX2 and p22 subunits, is responsible for transferring electrons from intracellular NADPH to extracellular oxygen1. This process generates superoxide anions that are vital for killing pathogens1. The activation of phagocyte NADPH oxidase requires membrane translocation and the binding of several cytosolic factors2. However, the exact mechanism by which cytosolic factors bind to and activate NOX2 is not well understood. Here we present the structure of the human NOX2-p22 complex activated by fragments of three cytosolic factors: p47, p67 and Rac1. The structure reveals that the p67-Rac1 complex clamps onto the dehydrogenase domain of NOX2 and induces its contraction, which stabilizes the binding of NADPH and results in a reduction of the distance between the NADPH-binding domain and the flavin adenine dinucleotide (FAD)-binding domain. Furthermore, the dehydrogenase domain docks onto the bottom of the transmembrane domain of NOX2, which reduces the distance between FAD and the inner haem. These structural rearrangements might facilitate the efficient transfer of electrons between the redox centres in NOX2 and lead to the activation of phagocyte NADPH oxidase.

Hepatitis C virus RNA is 5'-capped with flavin adenine dinucleotide.

RNA viruses have evolved elaborate strategies to protect their genomes, including 5' capping. However, until now no RNA 5' cap has been identified for hepatitis C virus1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer3. Here we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as a non-canonical initiating nucleotide by the viral RNA-dependent RNA polymerase, resulting in a 5'-FAD cap on the HCV RNA. The HCV FAD-capping frequency is around 75%, which is the highest observed for any RNA metabolite cap across all kingdoms of life4-8. FAD capping is conserved among HCV isolates for the replication-intermediate negative strand and partially for the positive strand. It is also observed in vivo on HCV RNA isolated from patient samples and from the liver and serum of a human liver chimeric mouse model. Furthermore, we show that 5'-FAD capping protects RNA from RIG-I mediated innate immune recognition but does not stabilize the HCV RNA. These results establish capping with cellular metabolites as a novel viral RNA-capping strategy, which could be used by other viruses and affect anti-viral treatment outcomes and persistence of infection.

Essential elements of radical pair magnetosensitivity in Drosophila.

Many animals use Earth's magnetic field (also known as the geomagnetic field) for navigation1. The favoured mechanism for magnetosensitivity involves a blue-light-activated electron-transfer reaction between flavin adenine dinucleotide (FAD) and a chain of tryptophan residues within the photoreceptor protein CRYPTOCHROME (CRY). The spin-state of the resultant radical pair, and therefore the concentration of CRY in its active state, is influenced by the geomagnetic field2. However, the canonical CRY-centric radical-pair mechanism does not explain many physiological and behavioural observations2-8. Here, using electrophysiology and behavioural analyses, we assay magnetic-field responses at the single-neuron and organismal levels. We show that the 52 C-terminal amino acid residues of Drosophila melanogaster CRY, lacking the canonical FAD-binding domain and tryptophan chain, are sufficient to facilitate magnetoreception. We also show that increasing intracellular FAD potentiates both blue-light-induced and magnetic-field-dependent effects on the activity mediated by the C terminus. High levels of FAD alone are sufficient to cause blue-light neuronal sensitivity and, notably, the potentiation of this response in the co-presence of a magnetic field. These results reveal the essential components of a primary magnetoreceptor in flies, providing strong evidence that non-canonical (that is, non-CRY-dependent) radical pairs can elicit magnetic-field responses in cells.

Complex II ambiguities-FADH2 in the electron transfer system.

The prevailing notion that reduced cofactors NADH and FADH2 transfer electrons from the tricarboxylic acid cycle to the mitochondrial electron transfer system creates ambiguities regarding respiratory Complex II (CII). CII is the only membrane-bound enzyme in the tricarboxylic acid cycle and is part of the electron transfer system of the mitochondrial inner membrane feeding electrons into the coenzyme Q-junction. The succinate dehydrogenase subunit SDHA of CII oxidizes succinate and reduces the covalently bound prosthetic group FAD to FADH2 in the canonical forward tricarboxylic acid cycle. However, several graphical representations of the electron transfer system depict FADH2 in the mitochondrial matrix as a substrate to be oxidized by CII. This leads to the false conclusion that FADH2 from the ß-oxidation cycle in fatty acid oxidation feeds electrons into CII. In reality, dehydrogenases of fatty acid oxidation channel electrons to the Q-junction but not through CII. The ambiguities surrounding Complex II in the literature and educational resources call for quality control, to secure scientific standards in current communications of bioenergetics, and ultimately support adequate clinical applications. This review aims to raise awareness of the inherent ambiguity crisis, complementing efforts to address the well-acknowledged issues of credibility and reproducibility.

The LOV-domain blue-light receptor LreA of the fungus Alternaria alternata binds predominantly FAD as chromophore and acts as a light and temperature sensor.

Light and temperature sensing are important features of many organisms. Light may provide energy but may also be used by non-photosynthetic organisms for orientation in the environment. Recent evidence suggests that plant and fungal phytochrome and plant phototropin serve dual functions as light and temperature sensors. Here we characterized the fungal LOV-domain blue-light receptor LreA of Alternaria alternata and show that it predominantly contains FAD as chromophore. Blue-light illumination induced ROS production followed by protein agglomeration in vitro. In vivo ROS may control LreA activity. LreA acts as a blue-light photoreceptor but also triggers temperature-shift-induced gene expression. Both responses required the conserved amino acid cysteine 421. We therefore propose that temperature mimics the photoresponse, which could be the ancient function of the chromoprotein. Temperature-dependent gene expression control with LreA was distinct from the response with phytochrome suggesting fine-tuned, photoreceptor-specific gene regulation.

RibU is an essential determinant of Listeria pathogenesis that mediates acquisition of FMN and FAD during intracellular growth.

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential riboflavin-derived cofactors involved in a myriad of redox reactions across all forms of life. Nevertheless, the basis of flavin acquisition strategies by riboflavin auxotrophic pathogens remains poorly defined. In this study, we examined how the facultative intracellular pathogen Listeria monocytogenes, a riboflavin auxotroph, acquires flavins during infection. A L. monocytogenes mutant lacking the putative riboflavin transporter (RibU) was completely avirulent in mice but had no detectable growth defect in nutrient-rich media. However, unlike wild type, the RibU mutant was unable to grow in defined media supplemented with FMN or FAD or to replicate in macrophages starved for riboflavin. Consistent with RibU functioning to scavenge FMN and FAD inside host cells, a mutant unable to convert riboflavin to FMN or FAD retained virulence and grew in cultured macrophages and in spleens and livers of infected mice. However, this FMN- and FAD-requiring strain was unable to grow in the gallbladder or intestines, where L. monocytogenes normally grows extracellularly, suggesting that these sites do not contain sufficient flavin cofactors to promote replication. Thus, by deleting genes required to synthesize FMN and FAD, we converted L. monocytogenes from a facultative to an obligate intracellular pathogen. Collectively, these data indicate that L. monocytogenes requires riboflavin to grow extracellularly in vivo but scavenges FMN and FAD to grow in host cells.

Ultrafast photooxidation of protein-bound anionic flavin radicals.

The photophysical properties of anionic semireduced flavin radicals are largely unknown despite their importance in numerous biochemical reactions. Here, we studied the photoproducts of these intrinsically unstable species in five different flavoprotein oxidases where they can be stabilized, including the well-characterized glucose oxidase. Using ultrafast absorption and fluorescence spectroscopy, we unexpectedly found that photoexcitation systematically results in the oxidation of protein-bound anionic flavin radicals on a time scale of less than ∼100 fs. The thus generated photoproducts decay back in the remarkably narrow 10- to 20-ps time range. Based on molecular dynamics and quantum mechanics computations, positively charged active-site histidine and arginine residues are proposed to be the electron acceptor candidates. Altogether, we established that, in addition to the commonly known and extensively studied photoreduction of oxidized flavins in flavoproteins, the reverse process (i.e., the photooxidation of anionic flavin radicals) can also occur. We propose that this process may constitute an excited-state deactivation pathway for protein-bound anionic flavin radicals in general. This hitherto undocumented photochemical reaction in flavoproteins further extends the family of flavin photocycles.

Elucidation of the Catalytic Sequence of Dihydroorotate Dehydrogenase B from Lactoccocus lactis: Evidence for Accumulation of a Flavin Bisemiquinone State in Catalysis.

The physiological role of dihydroorotate dehydrogenase (DHOD) enzymes is to catalyze the oxidation of dihydroorotate to orotate in pyrimidine biosynthesis. DHOD enzymes are structurally diverse existing as both soluble and membrane-associated forms. The Family 1 enzymes are soluble and act either as conventional single subunit flavin-dependent dehydrogenases known as Class 1A (DHODA) or as unusual heterodimeric enzymes known as Class 1B (DHODB). DHODBs possess two active sites separated by ∼20 Å, each with a noncovalently bound flavin cofactor. NAD is thought to interact at the FAD containing site, and the pyrimidine substrate is known to bind at the FMN containing site. At the approximate center of the protein is a single Fe2S2 center that is assumed to act as a conduit, facilitating one-electron transfers between the flavins. We present anaerobic transient state analysis of a DHODB enzyme from Lactoccocus lactis. The data presented primarily report the exothermic reaction that reduces orotate to dihydroorotate. The reductive half reaction reveals rapid two-electron reduction that is followed by the accumulation of a four-electron reduced state when NADH is added in excess, suggesting that the initial two electrons acquired reside on the FMN cofactor. Concomitant with the first reduction is the accumulation of a long-wavelength absorption feature consistent with the blue form of a flavin semiquinone. Spectral deconvolution and fitting to a model that includes reversibility for the second electron transfer reveals equilibrium accumulation of a flavin bisemiquinone state that has features of both red and blue semiquinones. Single turnover reactions with limiting NADH and excess orotate reveal that the flavin bisemiquinone accumulates with reduction of the enzyme by NADH and decays with reduction of the pyrimidine substrate, establishing the bisemiquinone as a fractional state of the two-electron reduced intermediate observed.

Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging.

Flavin-binding fluorescent proteins are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide, and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of 22 finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans. We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging of bacteria, as well as two-color imaging of mammalian cells (HEK293T), using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of flavin-binding fluorescent proteins in fluorescence microscopy.

Noncovalent interactions that tune the reactivities of the flavins in bifurcating electron transferring flavoprotein.

Bifurcating electron transferring flavoproteins (Bf-ETFs) tune chemically identical flavins to two contrasting roles. To understand how, we used hybrid quantum mechanical molecular mechanical calculations to characterize noncovalent interactions applied to each flavin by the protein. Our computations replicated the differences between the reactivities of the flavins: the electron transferring flavin (ETflavin) was calculated to stabilize anionic semiquinone (ASQ) as needed to execute its single-electron transfers, whereas the Bf flavin (Bfflavin) was found to disfavor the ASQ state more than does free flavin and to be less susceptible to reduction. The stability of ETflavin ASQ was attributed in part to H-bond donation to the flavin O2 from a nearby His side chain, via comparison of models employing different tautomers of His. This H-bond between O2 and the ET site was uniquely strong in the ASQ state, whereas reduction of ETflavin to the anionic hydroquinone (AHQ) was associated with side chain reorientation, backbone displacement, and reorganization of its H-bond network including a Tyr from the other domain and subunit of the ETF. The Bf site was less responsive overall, but formation of the Bfflavin AHQ allowed a nearby Arg side chain to adopt an alternative rotamer that can H-bond to the Bfflavin O4. This would stabilize the anionic Bfflavin and rationalize effects of mutation at this position. Thus, our computations provide insights on states and conformations that have not been possible to characterize experimentally, offering explanations for observed residue conservation and raising possibilities that can now be tested.

Uncovering Zn2+ as a cofactor of FAD-dependent Pseudomonas aeruginosa PAO1 d-2-hydroxyglutarate dehydrogenase.

Pseudomonas aeruginosa couples the oxidation of d-2-hydroxyglutarate (D2HG) to l-serine biosynthesis for survival, using d-2-hydroxyglutarate dehydrogenase from P. aeruginosa (PaD2HGDH). Knockout of PaD2HGDH impedes P. aeruginosa growth, making PaD2HGDH a potential target for therapeutics. Previous studies showed that the enzyme's activity increased with Zn2+, Co2+, or Mn2+ but did not establish the enzyme's metal composition and whether the metal is an activator or a required cofactor for the enzyme, which we addressed in this study. Comparable to the human enzyme, PaD2HGDH showed only 15% flavin reduction with D2HG or d-malate. Upon purifying PaD2HGDH with 1 mM Zn2+, the Zn2+:protein stoichiometry was 2:1, yielding an enzyme with ∼40 s-1kcat for d-malate. Treatment with 1 mM EDTA decreased the Zn2+:protein ratio to 1:1 without changing the kinetic parameters with d-malate. We observed complete enzyme inactivation for the metalloapoenzyme with 100 mM EDTA treatment, suggesting that Zn2+ is essential for PaD2HGDH activity. The presence of Zn2+ increased the flavin N3 atom pKa value to 11.9, decreased the flavin ε450 at pH 7.4 from 13.5 to 11.8 mM-1 cm-1, and yielded a charged transfer complex with a broad absorbance band >550 nm, consistent with a Zn2+-hydrate species altering the electronic properties of the enzyme-bound FAD. The exogenous addition of Zn2+, Co2+, Cd2+, Mn2+, or Ni2+ to the metalloapoenzyme reactivated the enzyme in a sigmoidal pattern, consistent with an induced fit rapid-rearrangement mechanism. Collectively, our data demonstrate that PaD2HGDH is a Zn2+-dependent metallo flavoprotein, which requires Zn2+ as an essential cofactor for enzyme activity.

The crystal structure of Vibrio cholerae (6-4) photolyase reveals interactions with cofactors and a DNA-binding region.

Photolyases (PLs) reverse UV-induced DNA damage using blue light as an energy source. Of these PLs, (6-4) PLs repair (6-4)-lesioned photoproducts. We recently identified a gene from Vibrio cholerae (Vc) encoding a (6-4) PL, but structural characterization is needed to elucidate specific interactions with the chromophore cofactors. Here, we determined the crystal structure of Vc (6-4) PL at 2.5 Å resolution. Our high-resolution structure revealed that the two well-known cofactors, flavin adenine dinucleotide and the photoantenna 6,7-dimethyl 8-ribityl-lumazin (DMRL), stably interact with an α-helical and an α/ß domain, respectively. Additionally, the structure has a third cofactor with distinct electron clouds corresponding to a [4Fe-4S] cluster. Moreover, we identified that Asp106 makes a hydrogen bond with water and DMRL, which indicates further stabilization of the photoantenna DMRL within Vc (6-4) PL. Further analysis of the Vc (6-4) PL structure revealed a possible region responsible for DNA binding. The region located between residues 478 to 484 may bind the lesioned DNA, with Arg483 potentially forming a salt bridge with DNA to stabilize further the interaction of Vc (6-4) PL with its substrate. Our comparative analysis revealed that the DNA lesion could not bind to the Vc (6-4) PL in a similar fashion to the Drosophila melanogaster (Dm, (6-4)) PL without a significant conformational change of the protein. The 23rd helix of the bacterial (6-4) PLs seems to have remarkable plasticity, and conformational changes facilitate DNA binding. In conclusion, our structure provides further insight into DNA repair by a (6-4) PL containing three cofactors.

A biosynthetic aspartate N-hydroxylase performs successive oxidations by holding intermediates at a site away from the catalytic center.

Nitrosuccinate is a biosynthetic building block in many microbial pathways. The metabolite is produced by dedicated L-aspartate hydroxylases that use NADPH and molecular oxygen as co-substrates. Here, we investigate the mechanism underlying the unusual ability of these enzymes to perform successive rounds of oxidative modifications. The crystal structure of Streptomyces sp. V2 L-aspartate N-hydroxylase outlines a characteristic helical domain wedged between two dinucleotide-binding domains. Together with NADPH and FAD, a cluster of conserved arginine residues forms the catalytic core at the domain interface. Aspartate is found to bind in an entry chamber that is close to but not in direct contact with the flavin. It is recognized by an extensive H-bond network that explains the enzyme's strict substrate-selectivity. A mutant designed to create steric and electrostatic hindrance to substrate binding disables hydroxylation without perturbing the NADPH oxidase side-activity. Critically, the distance between the FAD and the substrate is far too long to afford N-hydroxylation by the C4a-hydroperoxyflavin intermediate whose formation is confirmed by our work. We conclude that the enzyme functions through a catch-and-release mechanism. L-aspartate slides into the catalytic center only when the hydroxylating apparatus is formed. It is then re-captured by the entry chamber where it waits for the next round of hydroxylation. By iterating these steps, the enzyme minimizes the leakage of incompletely oxygenated products and ensures that the reaction carries on until nitrosuccinate is formed. This unstable product can then be engaged by a successive biosynthetic enzyme or undergoes spontaneous decarboxylation to produce 3-nitropropionate, a mycotoxin.

Metabolic response of auditory brainstem neurons to their broad physiological activity range.

Neurons exhibit a high energetic need, and the question arises as how they metabolically adapt to changing activity states. This is relevant for interpreting functional neuroimaging in different brain areas. Particularly, neurons with a broad firing range might exhibit metabolic adaptations. Therefore, we studied MNTB (medial nucleus of the trapezoid body) principal neurons, which generate action potentials (APs) at frequencies up to several hundred hertz. We performed the experiments in acute brainstem slices of the Mongolian gerbil (Meriones unguiculatus) at 22.5-24.5°C. Upon electrical stimulation of afferent MNTB fibres with 400 stimuli at varying frequencies, we monitored autofluorescence levels of NAD(P)H and FAD and determined the extremum amplitudes of their biphasic response. Additionally, we compared these data with alterations in O2 concentrations measured with an electrochemical sensor. These O2 changes are prominent since MNTB neurons rely on oxidative phosphorylation as shown by our pharmacological experiments. We calculated the O2 consumption rate as change in O2 concentration divided by stimulus durations, because these periods varied inversely with stimulus frequency as a result of the constant number of 400 stimuli applied. The O2 consumption rate increased with stimulation frequency up to a constant value at 600 Hz; that is, energy demand depends on temporal characteristics of activity despite the same number of stimuli. The rates showed no correlation with peak amplitudes of NAD(P)H or FAD, whilst the values of the two molecules were linearly correlated. This points at the complexity of analysing autofluorescence imaging for quantitative metabolic studies, because these values report only relative net changes of many superimposed oxidative and reductive processes. Monitoring O2 concentration rates is, thus, an important tool to improve the interpretation of NAD(P)H/FAD autofluorescence data, as they do not under all conditions and in all systems appropriately reflect the metabolic activity or energy demand.

Multispectral Imaging of Metabolic Fluorophores: Comparing In Vivo and Fresh Ex Vivo Tissue.

The enhanced uptake of glucose by cancer cells via aerobic glycolysis occurs when the lactic acid pathway is favored over the citric acid cycle. The lactic acid cycle in cancer cells influences the cytosolic concentration of metabolic fluorophores including NADH (the reduced form of nicotinamide adenine dinucleotide) and flavin adenine dinucleotide (FAD). In particular, the literature has shown that breast cancer influences the relative magnitude of fluorescence from NADH and FAD. A multispectral imaging system has been developed for rapid non-destructive imaging of intrinsic fluorescence in tissue. This paper compares in vivo data to fresh ex vivo data gathered as a function of time in mouse models. The data indicate that, if measured within 30 min of excision, a cancer diagnosis in fresh ex vivo tissue correlates with a cancer diagnosis in in vivo tissue. These results justify a plan to evaluate fresh ex vivo human tissue to quantify the sensitivity and specificity of the multispectral system.

Fus-SMO: Kinetics, Biochemical Characterisation and In Silico Modelling of a Chimeric Styrene Monooxygenase Demonstrating Quantitative Coupling Efficiency.

The styrene monooxygenase, a two-component enzymatic system for styrene epoxidation, was characterised through the study of Fus-SMO - a chimera resulting from the fusion of StyA and StyB using a flexible linker. Notably, it remains debated whether the transfer of FADH2 from StyB to StyA occurs through diffusion, channeling, or a combination of both. Fus-SMO was identified as a trimer with one bound FAD molecule. In silico modelling revealed a well-distanced arrangement (45-50 Å) facilitated by the flexible linker's loopy structure. Pre-steady-state kinetics elucidated the FADox reduction intricacies (kred=110 s-1 for bound FADox), identifying free FADox binding as the rate-determining step. The aerobic oxidation of FADH2 (kox=90 s-1) and subsequent decomposition to FADox and H2O2 demonstrated StyA's protective effect on the bound hydroperoxoflavin (kdec=0.2 s-1) compared to free cofactor (kdec=1.8 s-1). At varied styrene concentrations, kox for FADH2 ranged from 80 to 120 s-1. Studies on NADH consumption vs. styrene epoxidation revealed Fus-SMO's ability to achieve quantitative coupling efficiency in solution, surpassing natural two-component SMOs. The results suggest that Fus-SMO exhibits enhanced FADH2 channelling between subunits. This work contributes to comprehending FADH2 transfer mechanisms in SMO and illustrates how protein fusion can elevate catalytic efficiency for biocatalytic applications.

Rossmann-toolbox: a deep learning-based protocol for the prediction and design of cofactor specificity in Rossmann fold proteins.

The Rossmann fold enzymes are involved in essential biochemical pathways such as nucleotide and amino acid metabolism. Their functioning relies on interaction with cofactors, small nucleoside-based compounds specifically recognized by a conserved ßαß motif shared by all Rossmann fold proteins. While Rossmann methyltransferases recognize only a single cofactor type, the S-adenosylmethionine, the oxidoreductases, depending on the family, bind nicotinamide (nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate) or flavin-based (flavin adenine dinucleotide) cofactors. In this study, we showed that despite its short length, the ßαß motif unambiguously defines the specificity towards the cofactor. Following this observation, we trained two complementary deep learning models for the prediction of the cofactor specificity based on the sequence and structural features of the ßαß motif. A benchmark on two independent test sets, one containing ßαß motifs bearing no resemblance to those of the training set, and the other comprising 38 experimentally confirmed cases of rational design of the cofactor specificity, revealed the nearly perfect performance of the two methods. The Rossmann-toolbox protocols can be accessed via the webserver at https://lbs.cent.uw.edu.pl/rossmann-toolbox and are available as a Python package at https://github.com/labstructbioinf/rossmann-toolbox.

Label-Free Optical Metabolic Imaging in Cells and Tissues.

Over the last half century, the autofluorescence of the metabolic cofactors NADH (reduced nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) has been quantified in a variety of cell types and disease states. With the spread of nonlinear optical microscopy techniques in biomedical research, NADH and FAD imaging has offered an attractive solution to noninvasively monitor cell and tissue status and elucidate dynamic changes in cell or tissue metabolism. Various tools and methods to measure the temporal, spectral, and spatial properties of NADH and FAD autofluorescence have been developed. Specifically, an optical redox ratio of cofactor fluorescence intensities and NADH fluorescence lifetime parameters have been used in numerous applications, but significant work remains to mature this technology for understanding dynamic changes in metabolism. This article describes the current understanding of our optical sensitivity to different metabolic pathways and highlights current challenges in the field. Recent progress in addressing these challenges and acquiring more quantitative information in faster and more metabolically relevant formats is also discussed.

A multifunctional flavoprotein monooxygenase HspB for hydroxylation and C-C cleavage of 6-hydroxy-3-succinoyl-pyridine.

Flavoprotein monooxygenases catalyze reactions, including hydroxylation and epoxidation, involved in the catabolism, detoxification, and biosynthesis of natural substrates and industrial contaminants. Among them, the 6-hydroxy-3-succinoyl-pyridine (HSP) monooxygenase (HspB) from Pseudomonas putida S16 facilitates the hydroxylation and C-C bond cleavage of the pyridine ring in nicotine. However, the mechanism for biodegradation remains elusive. Here, we refined the crystal structure of HspB and elucidated the detailed mechanism behind the oxidative hydroxylation and C-C cleavage processes. Leveraging structural information about domains for binding the cofactor flavin adenine dinucleotide (FAD) and HSP substrate, we used molecular dynamics simulations and quantum/molecular mechanics calculations to demonstrate that the transfer of an oxygen atom from the reactive FAD peroxide species (C4a-hydroperoxyflavin) to the C3 atom in the HSP substrate constitutes a rate-limiting step, with a calculated reaction barrier of about 20 kcal/mol. Subsequently, the hydrogen atom was rebounded to the FAD cofactor, forming C4a-hydroxyflavin. The residue Cys218 then catalyzed the subsequent hydrolytic process of C-C cleavage. Our findings contribute to a deeper understanding of the versatile functions of flavoproteins in the natural transformation of pyridine and HspB in nicotine degradation.IMPORTANCEPseudomonas putida S16 plays a pivotal role in degrading nicotine, a toxic pyridine derivative that poses significant environmental challenges. This study highlights a key enzyme, HspB (6-hydroxy-3-succinoyl-pyridine monooxygenase), in breaking down nicotine through the pyrrolidine pathway. Utilizing dioxygen and a flavin adenine dinucleotide cofactor, HspB hydroxylates and cleaves the substrate's side chain. Structural analysis of the refined HspB crystal structure, combined with state-of-the-art computations, reveals its distinctive mechanism. The crucial function of Cys218 was never discovered in its homologous enzymes. Our findings not only deepen our understanding of bacterial nicotine degradation but also open avenues for applications in both environmental cleanup and pharmaceutical development.