Discovery of a new flavin N5-adduct in a tyrosine to phenylalanine variant of d-Arginine dehydrogenase.

d-Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) catalyzes the flavin-dependent oxidation of d-arginine and other d-amino acids. Here, we report the crystal structure at 1.29 Å resolution for PaDADH-Y249F expressed and co-crystallized with d-arginine. The overall structure of PaDADH-Y249F resembled PaDADH-WT, but the electron density for the flavin cofactor was ambiguous, suggesting the presence of modified flavins. Electron density maps and mass spectrometric analysis confirmed the presence of both N5-(4-guanidino-oxobutyl)-FAD and 6-OH-FAD in a single crystal of PaDADH-Y249F and helped with the further refinement of the X-ray crystal structure. The versatility of the reduced flavin is apparent in the PaDADH-Y249F structure and is evidenced by the multiple functions it can perform in the same active site.

Plant cell wall architecture guided design of CBM3-GH11 chimeras with enhanced xylanase activity using a tandem repeat left-handed ß-3-prism scaffold.

Effective use of plant biomass as an abundant and renewable feedstock for biofuel production and biorefinery requires efficient enzymatic mobilization of cell wall polymers. Knowledge of plant cell wall composition and architecture has been exploited to develop novel multifunctional enzymes with improved activity against lignocellulose, where a left-handed ß-3-prism synthetic scaffold (BeSS) was designed for insertion of multiple protein domains at the prism vertices. This allowed construction of a series of chimeras fusing variable numbers of a GH11 ß-endo-1,4-xylanase and the CipA-CBM3 with defined distances and constrained relative orientations between catalytic domains. The cellulose binding and endoxylanase activities of all chimeras were maintained. Activity against lignocellulose substrates revealed a rapid 1.6- to 3-fold increase in total reducing saccharide release and increased levels of all major oligosaccharides as measured by polysaccharide analysis using carbohydrate gel electrophoresis (PACE). A construct with CBM3 and GH11 domains inserted in the same prism vertex showed highest activity, demonstrating interdomain geometry rather than number of catalytic sites is important for optimized chimera design. These results confirm that the BeSS concept is robust and can be successfully applied to the construction of multifunctional chimeras, which expands the possibilities for knowledge-based protein design.

A Single-Point Mutation in d-Arginine Dehydrogenase Unlocks a Transient Conformational State Resulting in Altered Cofactor Reactivity.

Proteins are inherently dynamic, and proper enzyme function relies on conformational flexibility. In this study, we demonstrated how an active site residue changes an enzyme's reactivity by modulating fluctuations between conformational states. Replacement of tyrosine 249 (Y249) with phenylalanine in the active site of the flavin-dependent d-arginine dehydrogenase yielded an enzyme with both an active yellow FAD (Y249F-y) and an inactive chemically modified green FAD, identified as 6-OH-FAD (Y249F-g) through various spectroscopic techniques. Structural investigation of Y249F-g and Y249F-y variants by comparison to the wild-type enzyme showed no differences in the overall protein structure and fold. A closer observation of the active site of the Y249F-y enzyme revealed an alternative conformation for some active site residues and the flavin cofactor. Molecular dynamics simulations probed the alternate conformations observed in the Y249F-y enzyme structure and showed that the enzyme variant with FAD samples a metastable conformational state, not available to the wild-type enzyme. Hybrid quantum/molecular mechanical calculations identified differences in flavin electronics between the wild type and the alternate conformation of the Y249F-y enzyme. The computational studies further indicated that the alternate conformation in the Y249F-y enzyme is responsible for the higher spin density at the C6 atom of flavin, which is consistent with the formation of 6-OH-FAD in the variant enzyme. The observations in this study are consistent with an alternate conformational space that results in fine-tuning the microenvironment around a versatile cofactor playing a critical role in enzyme function.

New frontiers in flavin-dependent monooxygenases.

Flavin-dependent monooxygenases catalyze a wide variety of redox reactions in important biological processes and are responsible for the synthesis of highly complex natural products. Although much has been learned about FMO chemistry in the last ~80 years of research, several aspects of the reactions catalyzed by these enzymes remain unknown. In this review, we summarize recent advancements in the flavin-dependent monooxygenase field including aspects of flavin dynamics, formation and stabilization of reactive species, and the hydroxylation mechanism. Novel catalysis of flavin-dependent N-oxidases involving consecutive oxidations of amines to generate oximes or nitrones is presented and the biological relevance of the products is discussed. In addition, the activity of some FMOs have been shown to be essential for the virulence of several human pathogens. We also discuss the biomedical relevance of FMOs in antibiotic resistance and the efforts to identify inhibitors against some members of this important and growing family enzymes.

Kinetic Investigation of a Presumed Nitronate Monooxygenase from Pseudomonas aeruginosa PAO1 Establishes a New Class of NAD(P)H:Quinone Reductases.

PA0660 from Pseudomonas aeruginosa PAO1 is currently classified as a hypothetical nitronate monooxygenase (NMO), but no evidence at the transcript or protein level has been presented. In this study, PA0660 was purified and its biochemical and kinetic properties were characterized. Absorption spectroscopy and mass spectrometry demonstrated a tightly, noncovalently bound FMN in the active site of the enzyme. Analytical ultracentrifugation showed that the enzyme exists as a dimer in solution. Despite its annotation, PA0660 did not exhibit nitronate monooxygenase activity. The enzyme could be reduced with NADPH or NADH with a marked preference for NADPH, as indicated by ∼30-fold larger kcat/ Km and kred/ Kd values. Turnover could be sustained with NAD(P)H and quinones, DCPIP, and to a lesser extent molecular oxygen. However, PA0660 did not turn over with methyl red, consistent with a lack of azoreductase activity. The enzyme turned over through a ping-pong bi-bi steady-state kinetic mechanism with NADPH and 1,4-benzoquinone showing a kcat value of 90 s-1. The rate constant for flavin reduction with saturating NADPH was 360 s-1, whereas that for flavin oxidation with 1,4-benzoquinone was 270 s-1, consistent with both hydride transfers from the pyridine nucleotide to the flavin and from the flavin to 1,4-benzoquinone being partially rate-limiting for enzyme turnover. A BlastP search and a multiple-sequence alignment analysis of PA0660 highlighted the presence of six conserved motifs in >1000 open reading frames currently annotated as hypothetical NMOs. Our results suggest that PA0660 should be classified as an NAD(P)H:quinone reductase and serve as a paradigm enzyme for a new class of enzymes.

Structural basis for the design of selective inhibitors for Schistosoma mansoni dihydroorotate dehydrogenase.

Trematode worms of the genus Schistosoma are the causing agents of schistosomiasis, a parasitic disease responsible for a considerable economic and healthy burden worldwide. In the present work, the characterization of the enzyme dihydroorotate dehydrogenase from Schistosoma mansoni (SmDHODH) is presented. Our studies demonstrated that SmDHODH is a member of class 2 DHODHs and catalyzes the oxidation of dihydroorotate into orotate using quinone as an electron acceptor by employing a ping-pong mechanism of catalysis. SmDHODH homology model showed the presence of all structural features reported for class 2 DHODH enzymes and reveal the presence of an additional protuberant domain predicted to fold as a flexible loop and absent in the other known class 2 DHODHs. Molecular dynamics simulations showed that the ligand-free forms of SmDHODH and HsDHODH undergo different rearrangements in solution. Well-known class 2 DHODH inhibitors were tested against SmDHODH and HsDHODH and the results suggest that the variable nature of the quinone-binding tunnel between human and parasite enzymes, as well as the differences in structural plasticity involving rearrangements of the N-terminal α-helical domain can be exploited for the design of SmDHODH selective inhibitors, as a strategy to validate DHODH as a drug target against schistosomiasis.

Steric hindrance controls pyridine nucleotide specificity of a flavin-dependent NADH:quinone oxidoreductase.

The crystal structure of the NADH:quinone oxidoreductase PA1024 has been solved in complex with NAD+ to 2.2 Å resolution. The nicotinamide C4 is 3.6 Å from the FMN N5 atom, with a suitable orientation for facile hydride transfer. NAD+ binds in a folded conformation at the interface of the TIM-barrel domain and the extended domain of the enzyme. Comparison of the enzyme-NAD+ structure with that of the ligand-free enzyme revealed a different conformation of a short loop (75-86) that is part of the NAD+ -binding pocket. P78, P82, and P84 provide internal rigidity to the loop, whereas Q80 serves as an active site latch that secures the NAD+ within the binding pocket. An interrupted helix consisting of two α-helices connected by a small three-residue loop binds the pyrophosphate moiety of NAD+ . The adenine moiety of NAD+ appears to π-π stack with Y261. Steric constraints between the adenosine ribose of NAD+ , P78, and Q80, control the strict specificity of the enzyme for NADH. Charged residues do not play a role in the specificity of PA1024 for the NADH substrate.

Crystal structure of yeast nitronate monooxygenase from Cyberlindnera saturnus.

Nitronate monooxygenase (NMO) is an FMN-dependent enzyme that oxidizes the neurotoxin propionate 3-nitronate (P3N) and represents the best-known system for P3N detoxification in different organisms. The crystal structure of the first eukaryotic Class I NMO from Cyberlindnera saturnus (CsNMO) has been solved at 1.65 Å resolution and refined to an R-factor of 14.0%. The three-dimensional structures of yeast CsNMO and bacterial PaNMO are highly conserved with the exception of three additional loops on the surface in the CsNMO enzyme and differences in four active sites residues. A PEG molecule was identified in the structure and formed extensive interactions with CsNMO, suggesting a specific binding site; however, 8% PEG showed no significant effect on the enzyme activity. This new crystal structure of a eukaryotic NMO provides insight into the function of this class of enzymes.

The dihydroorotate dehydrogenases: Past and present.

The flavoenzyme dihydroorotate dehydrogenase catalyzes the stereoselective oxidation of (S)-dihydroorotate to orotate in the fourth of the six conserved enzymatic reactions involved in the de novo pyrimidine biosynthetic pathway. Inhibition of pyrimidine metabolism by selectively targeting DHODHs has been exploited in the development of new therapies against cancer, immunological disorders, bacterial and viral infections, and parasitic diseases. Through a chronological narrative, this review summarizes the efforts of the scientific community to achieve our current understanding of structural and biochemical properties of DHODHs. It also attempts to describe the latest advances in medicinal chemistry for therapeutic development based on the selective inhibition of DHODH, including an overview of the experimental techniques used for ligand screening during the process of drug discovery.

Amine oxidation by d-arginine dehydrogenase in Pseudomonas aeruginosa.

d-Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) is a flavin-dependent oxidoreductase, which is part of a novel two-enzyme racemization system that functions to convert d-arginine to l-arginine. PaDADH contains a noncovalently linked FAD that shows the highest activity with d-arginine. The enzyme exhibits broad substrate specificity towards d-amino acids, particularly with cationic and hydrophobic d-amino acids. Biochemical studies have established the structure and the mechanistic properties of the enzyme. The enzyme is a true dehydrogenase because it displays no reactivity towards molecular oxygen. As established through solvent and multiple kinetic isotope studies, PaDADH catalyzes an asynchronous CH and NH bond cleavage via a hydride transfer mechanism. Steady-state kinetic studies with d-arginine and d-histidine are consistent with the enzyme following a ping-pong bi-bi mechanism. As shown by a combination of crystallography, kinetic and computational data, the shape and flexibility of loop L1 in the active site of PaDADH are important for substrate capture and broad substrate specificity.

The mechanistic study of Leishmania major dihydro-orotate dehydrogenase based on steady- and pre-steady-state kinetic analysis.

Leishmania major dihydro-orotate dehydrogenase (DHODHLm) has been considered as a potential therapeutic target against leishmaniasis. DHODHLm, a member of class 1A DHODH, oxidizes dihydro-orotate (DHO) to orotate (ORO) during pyrimidine biosynthesis using fumarate (FUM) as the oxidizing substrate. In the present study, the chemistry of reduction and reoxidation of the flavin mononucleotide (FMN) cofactor in DHODHLm was examined by steady- and pre-steady state kinetics under both aerobic and anaerobic environments. Our results provide for the first time the experimental evidence of co-operative behaviour in class 1A DHODH regulated by DHO binding and reveal that the initial reductive flavin half-reaction follows a mechanism with two steps. The first step is consistent with FMN reduction and shows a hyperbolic dependence on the DHO concentration with a limiting rate (kred) of 110±6 s(-1) and a K(DHO) d of 180±27 µM. Dissociation of the reduced flavin-ORO complex corresponds to the second step, with a limiting rate of 6 s(-1). In the oxidative half-reaction, the oxygen-sensitive reoxidation of the reduced FMN cofactor of DHODHLm by FUM exhibited a hyperbolic saturation profile dependent on FUM concentration allowing estimation of K(FUM) d and the limiting rate (kreox) of 258±53 µM and 35±2 s(-1), respectively. Comparison between steady- and pre-steady-state parameters together with studies of interaction for DHODHLm with both ORO and succinate (SUC), suggests that ORO release is the rate-limiting step in overall catalysis. Our results provide evidence of mechanistic differences between class 1A and class 2 individual half-reactions to be exploited for the development of selective inhibitors.