Mechanistic Insights into the N-Hydroxylations Catalyzed by the Binuclear Iron Domain of SznF Enzyme: Key Piece in the Synthesis of Streptozotocin.

SznF, a member of the emerging family of heme-oxygenase-like (HO-like) di-iron oxidases and oxygenases, employs two distinct domains to catalyze the conversion of Nω-methyl-L-arginine (L-NMA) into N-nitroso-containing product, which can subsequently be transformed into streptozotocin. Using unrestricted density functional theory (UDFT) with the hybrid functional B3LYP, we have mechanistically investigated the two sequential hydroxylations of L-NMA catalyzed by SznF's binuclear iron central domain. Mechanism B primarily involves the O-O bond dissociation, forming Fe(IV)=O, induced by the H+/e- introduction to the FeA side of µ-1,2-peroxo-Fe2(III/III), the substrate hydrogen abstraction by Fe(IV)=O, and the hydroxyl rebound to the substrate N radical. The stochastic addition of H+/e- to the FeB side (mechanism C) can transition to mechanism B, thereby preventing enzyme deactivation. Two other competing mechanisms, involving the direct O-O bond dissociation (mechanism A) and the addition of H2O as a co-substrate (mechanism D), have been ruled out.

A comparative study of N-hydroxylating flavoprotein monooxygenases reveals differences in kinetics and cofactor binding.

Many natural products comprise N-O containing functional groups with crucial roles for biological activity. Their enzymatic formation is predominantly achieved by oxidation of an amine to form a hydroxylamine, which enables further functionalization. N-hydroxylation by flavin-dependent enzymes has so far been attributed to a distinct group of flavoprotein monooxygenases (FPMOs) containing two dinucleotide binding domains. Here, we present three flavoprotein N-hydroxylases that exhibit a glutathione reductase 2 (GR2)-type topology with only one nucleotide binding domain, which belong to a distinct phylogenetic branch within the GR2-fold FPMOs. In addition to PqsL of Pseudomonas aeruginosa, which catalyses the N-hydroxylation of a primary aromatic amine during biosynthesis of 2-alkyl-4-hydroxyquinoline N-oxide respiratory chain inhibitors, we analysed isofunctional orthologs from Burkholderia thailandensis (HmqL) and Chryseobacterium nematophagum (PqsLCn ). Pre-steady-state kinetics revealed that the oxidative half-reaction of all three enzymes is highly efficient despite the soft nucleophile substrate. Ligand binding studies indicated that HmqL and PqsLCn show displacement of the oxidized flavin cofactor from the active site by the organic substrate, which likely abolishes the substrate inhibition observed in PqsL. Despite mechanistic heterogeneity, the investigated monooxygenases in principle follow the catalytic mechanism of GR2-fold FPMOs and thus differ from previously described N-hydroxylating enzymes. The discovery of this yet unrecognized family of flavoprotein N-hydroxylases expands the current knowledge on the catalytic repertoire of GR2-type FPMOs and provides a basis for the discovery of other nitrogen functionalizing reactions.

A flavin-dependent monooxygenase produces nitrogenous tomato aroma volatiles using cysteine as a nitrogen source.

Tomato (Solanum lycopersicum) produces a wide range of volatile chemicals during fruit ripening, generating a distinct aroma and contributing to the overall flavor. Among these volatiles are several aromatic and aliphatic nitrogen-containing compounds for which the biosynthetic pathways are not known. While nitrogenous volatiles are abundant in tomato fruit, their content in fruits of the closely related species of the tomato clade is highly variable. For example, the green-fruited species Solanum pennellii are nearly devoid, while the red-fruited species S. lycopersicum and Solanum pimpinellifolium accumulate high amounts. Using an introgression population derived from S. pennellii, we identified a locus essential for the production of all the detectable nitrogenous volatiles in tomato fruit. Silencing of the underlying gene (SlTNH1;Solyc12g013690) in transgenic plants abolished production of aliphatic and aromatic nitrogenous volatiles in ripe fruit, and metabolomic analysis of these fruit revealed the accumulation of 2-isobutyl-tetrahydrothiazolidine-4-carboxylic acid, a known conjugate of cysteine and 3-methylbutanal. Biosynthetic incorporation of stable isotope-labeled precursors into 2-isobutylthiazole and 2-phenylacetonitrile confirmed that cysteine provides the nitrogen atom for all nitrogenous volatiles in tomato fruit. Nicotiana benthamiana plants expressing SlTNH1 readily transformed synthetic 2-substituted tetrahydrothiazolidine-4-carboxylic acid substrates into a mixture of the corresponding 2-substituted oxime, nitro, and nitrile volatiles. Distinct from other known flavin-dependent monooxygenase enzymes in plants, this tetrahydrothiazolidine-4-carboxylic acid N-hydroxylase catalyzes sequential hydroxylations. Elucidation of this pathway is a major step forward in understanding and ultimately improving tomato flavor quality.

Differences in Pharmacokinetics and Haematotoxicities of Aniline and Its Dimethyl Derivatives Orally Administered in Rats.

Aniline and its dimethyl derivatives reportedly become haematotoxic after metabolic N-hydroxylation of their amino groups. The plasma concentrations of aniline and its dimethyl derivatives after single oral doses of 25 mg/kg in rats were quantitatively measured and semi-quantitatively estimated using LC-tandem mass spectrometry. The quantitatively determined elimination rates of aniline; 2,4-dimethylaniline; and 3,5-dimethylaniline based on rat plasma versus time curves were generally rapid compared with those of 2,3-; 2,5-; 2,6-; and N,2-dimethylaniline. The primary acetylated metabolites of aniline; 2,4-dimethylaniline; and 3,5-dimethylaniline, as semi-quantitatively estimated based on their peak areas in LC analyses, were more extensively formed than those of 2,3-; 2,5-; 2,6-; and N,2-dimethylaniline. The areas under the curve of unmetabolized (remaining) aniline and its dimethyl derivatives estimated using simplified physiologically based pharmacokinetic models (that were set up using the experimental plasma concentrations) showed an apparently positive correlation with the reported lowest-observed-effect levels for haematotoxicity of these chemicals. In the case of 2,4-dimethylaniline, a methyl group at another C4-positon would be one of the determinant factors for rapid metabolic elimination to form aminotoluic acid. These results suggest that rapid and extensive metabolic activation of aniline and its dimethyl derivatives occurred in rats and that the presence of a methyl group at the C2-positon may generally suppress fast metabolic rates of dimethyl aniline derivatives that promote metabolic activation reactions at NH2 moieties.

Dapsone-induced hepatic complications: it's time to think beyond methemoglobinemia.

Drug-induced liver injury is an important cause of hepatotoxicity and poses a challenging clinical problem with respect to both diagnosis and management. Patients susceptible to hepatotoxicity on exposure to dapsone is constantly on the rise. Dapsone (4,4'-diaminodiphenylsulfone) is clinically used alone or in combination with rifampicin for the treatment of a variety of dermatological disorders such as acne, dermatitis herpetiformis, psoriasis, Toxoplasma gondii infections, leprosy and pneumocystis carinii pneumonia in AIDS patients. However, the clinical use of dapsone is limited because of dose-dependent adverse hematological reactions. The cholestatic injury caused by dapsone and its N- hydroxylated metabolites hinders bile flow and causes oxidative stress and hepatic necrosis, further, leading to hemolysis responsible for hepatitis due to iron overload in the liver. Hence, clinicians' awareness of the hepatotoxic potential of dapsone is highly warranted.

l-lysine metabolism to N-hydroxypipecolic acid: an integral immune-activating pathway in plants.

l-lysine catabolic routes in plants include the saccharopine pathway to α-aminoadipate and decarboxylation of lysine to cadaverine. The current review will cover a third l-lysine metabolic pathway having a major role in plant systemic acquired resistance (SAR) to pathogen infection that was recently discovered in Arabidopsis thaliana. In this pathway, the aminotransferase AGD2-like defense response protein (ALD1) α-transaminates l-lysine and generates cyclic dehydropipecolic (DP) intermediates that are subsequently reduced to pipecolic acid (Pip) by the reductase SAR-deficient 4 (SARD4). l-pipecolic acid, which occurs ubiquitously in the plant kingdom, is further N-hydroxylated to the systemic acquired resistance (SAR)-activating metabolite N-hydroxypipecolic acid (NHP) by flavin-dependent monooxygenase1 (FMO1). N-hydroxypipecolic acid induces the expression of a set of major plant immune genes to enhance defense readiness, amplifies resistance responses, acts synergistically with the defense hormone salicylic acid, promotes the hypersensitive cell death response and primes plants for effective immune mobilization in cases of future pathogen challenge. This pathogen-inducible NHP biosynthetic pathway is activated at the transcriptional level and involves feedback amplification. Apart from FMO1, some cytochrome P450 monooxygenases involved in secondary metabolism catalyze N-hydroxylation reactions in plants. In specific taxa, pipecolic acid might also serve as a precursor in the biosynthesis of specialized natural products, leading to C-hydroxylated and otherwise modified piperidine derivatives, including indolizidine alkaloids. Finally, we show that NHP is glycosylated in Arabidopsis to form a hexose-conjugate, and then discuss open questions in Pip/NHP-related research.