Phenylalanine hydroxylase is a mononuclear non-heme iron protein that uses tetrahydropterin as the source of the two electrons needed to activate dioxygen for the hydroxylation of phenylalanine to tyrosine. Rapid-quench methods have been used to analyze the mechanism of a bacterial phenylalanine hydroxylase from Chromobacterium violaceum. Mössbauer spectra of samples prepared by freeze-quenching the reaction of the enzyme/57Fe(II)/phenylalanine/6-methyltetrahydropterin complex with O2 reveal the accumulation of an intermediate at short reaction times (20–100 ms). The Mössbauer parameters of the intermediate, δ = 0.28 mm/s and |ΔEQ| = 1.26 mm/s, suggest that it is a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(II) hydroxylases, including a tetrahydropterin-dependent tyrosine hydroxylase. Analysis of the tyrosine content of acid-quenched samples from similar reactions establishes that the Fe(IV) intermediate is kinetically competent to be the hydroxylating intermediate. Similar chemical-quench analysis of a reaction allowed to proceed for several turnovers shows a burst of tyrosine formation, consistent with rate-limiting product release. All three data sets can be modeled with a mechanism in which the enzyme-substrate complex reacts with oxygen to form an Fe(IV)=O intermediate with a rate constant of 19 mM−1s−1, the Fe(IV)=O hydroxylates phenylalanine with a rate constant of 42 s−1, and rate-limiting product release occurs at 6 s−1 at 5°C.

Phenylalanine hydroxylase from Chromobacterium violaceum (CvPheH) is a non-heme iron monooxygenase that catalyzes the hydroxylation of phenylalanine to tyrosine. In the present study we used deuterium kinetic isotope effects to probe the chemical mechanisms of aromatic and benzylic hydroxylation in order to compare the reactivities of bacterial and eukaryotic aromatic amino acid hydroxylases. The D k cat value for the reaction of CvPheH with 2 H 5 -phenylalanine is 1.2 with 6-methyltetrahydropterin and 1.4 with 6,7-dimethyltetrahydropterin. With the mutant enzyme I234D, the D k cat value decreases to 0.9 with the latter pterin; this is likely to be the intrinsic effect for oxygen addition to the amino acid. The isotope effect on the subsequent tautomerization of a dienone intermediate was determined to be 5.1 by measuring the retention of deuterium in tyrosine produced from partially deuterated phenylalanine; this large isotope effect is responsible for the normal effect on k cat . The isotope effect for hydroxylation of the methyl group of 4-CH 3 -phenylalanine, obtained from the partitioning of benzylic and aromatic hydroxylation products, is 10. The temperature dependence of this isotope effect establishes the contribution of hydrogen tunneling to benzylic hydroxylation by this enzyme. The results presented here provide evidence that the reactivities of the prokaryotic and eukaryotic hydroxylases are similar and further define the reactivity of the iron center for the family of aromatic amino acid hydroxylases.Phenylalanine hydroxylase (PheH) 1 is a non-heme iron monooxygenase that catalyzes the hydroxylation of phenylalanine to form tyrosine (Scheme 1) (1). In humans, the enzyme is responsible for catabolism of excess phenylalanine in the diet, and mutations in PheH result in the metabolic disorder phenylketonuria (2). In addition, over 150 bacterial genomes have been reported to include a gene for PheH. The phenylalanine hydroxylase from Chromobacterium violaceum (CvPheH) has been the most studied, having been cloned and expressed in Escherichia coli (3,4). Scheme 2 shows the present understanding of the mechanism of aromatic amino acid hydroxylation based on studies of the eukaryotic PheH and the other two aromatic amino acid hydroxylases, tyrosine hydroxylase (TyrH) and tryptophan hydroxylase (5). After the three substrates are bound, molecular oxygen forms a peroxo bridge between the 4a position of the pterin and the iron. The oxygen-oxygen bond then cleaves to form the Fe(IV)O hydroxylating species and the 4a-hydroxypterin product (6-8). Direct † This work was supported in part by grants from the NIH (GM47291) and The Welch Foundation (A-1245).*Address correspondence to: Paul F. Fitzpatrick, Department of Biochemistry and Biophysics, 2128 TAMU, College Station, TX 77843-2128, Ph: 979-845-5487, Fax: 979-845-4946, Email: fitzpat@tamu.edu. 1 Abbreviations: PheH, phenylalanine hydroxylase; CvPheH, Chromobacterium violaceum phenylalanine hydroxylase; TyrH, tyrosine hydroxylase; 6-MePH 4 , 6-methylte...

We report the full reduction of the biological cofactor FMN with visible light using CdSe quantum dots and methylviologen as an electron relay. In turn, these reducing equivalents can drive the stereospecific reduction of ketoisophorone by an old yellow enzyme homologue from Bacillus subtilis (YqjM). The experiments demonstrate the current capabilities and limitations of quantum dots as part of a cofactor regeneration system and pave the road for future studies aimed at new and improved in situ light-driven cofactor regeneration strategies

Plant extracts from the genus Cecropia have been used by Latin-American traditional medicine to treat metabolic disorders and diabetes. Previous results have shown that roots of Cecropia telenitida contain pentacyclic triterpenes and these molecules display a hypoglycemic effect in an insulin-resistant murine model. The pharmacological target of these molecules, however, remains unknown. Several lines of evidence indicate that pentacyclic triterpenes inhibit the 11β-hydroxysteroid dehydrogenase type 1 enzyme, which highlights the potential use of this type of natural product as phytotherapeutic or botanical dietary supplements. The main goal of the study was the evaluation of the inhibitory effect of Cecropia telenitida molecules on 11β-hydroxysteroid dehydrogenase type 1 enzyme activity. A pre-fractionated chemical library was obtained from the roots of Cecropia telenitida using several automated chromatography separation steps and a homogeneous time resolved fluorescence assay was used for the bio-guided isolation of inhibiting molecules. The screening of a chemical library consisting of 125 chemical purified fractions obtained from Cecropia telenitida roots identified one fraction displaying 82% inhibition of the formation of cortisol by the 11β-hydroxysteroid dehydrogenase type 1 enzyme. Furthermore, a molecule displaying IC50 of 0.95 ± 0.09 µM was isolated from this purified fraction and structurally characterized, which confirms that a pentacyclic triterpene scaffold was responsible for the observed inhibition. Our results support the hypothesis that pentacyclic triterpene molecules from Cecropia telenitida can inhibit 11β-hydroxysteroid dehydrogenase type 1 enzyme activity. These findings highlight the potential ethnopharmacological use of plants from the genus Cecropia for the treatment of metabolic disorders and diabetes.

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Promising research over the past decades has shown that some types of pentacyclic triterpenes (PTs) are associated with the prevention of type 2 diabetes (T2D), especially those found in foods. The most abundant edible sources of PTs are those belonging to the ursane and oleanane scaffold. The principal finding is that Cecropia telenitida contains abundant oleanane and ursane PT types with similar oxygenation patterns to those found in food matrices. We studied the compositional profile of a rich PT fraction (DE16-R) and carried out a viability test over different cell lines. The biosynthetic pathway connected to the isolated PTs in C.telenitida offers a specific medicinal benefit related to the modulation of T2D. This current study suggests that this plant can assemble isobaric, positional isomers or epimeric PT. Ursane or oleanane scaffolds with the same oxygenation pattern are always shared by the PTs in C. telenitida, as demonstrated by its biosynthetic pathway. Local communities have long used this plant in traditional medicine, and humans have consumed ursane and oleanane PTs in fruits since ancient times, two key points we believe useful in considering the medicinal benefits of C. telenitida and explaining how a group of molecules sharing a closely related scaffold can express effectiveness.

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The non-heme iron enzyme phenylalanine hydroxylase from Chromobacterium violaceum has previously been shown to catalyze the hydroxylation of benzylic and aliphatic carbons in addition to the normal aromatic hydroxylation reaction. The intrinsic isotope effect for hydroxylation of 3-cyclochexylalanine by the enzyme was determined to gain insight into the reactivity of the iron center. With 3-[ 2 H 11 -cyclohexyl]-alanine as substrate, the isotope effect on the k cat value was 1, consistent with an additional step in the overall reaction being significantly slower than hydroxylation. Consequently the isotope effect was determined as an intramolecular effect, measuring the amount of deuterium lost in the hydroxylation of 3-[1,2,3,4,5,6-2 H 6 -cyclohexyl]-alanine. The ratio of 4-HO-cyclohexylalanine that retained deuterium to that which lost one deuterium atom was 2.8. This allowed calculation of 12.6 as the ratio of the primary deuterium kinetic isotope effect to the secondary isotope effect. This value is consistent with hydrogen atom abstraction by an electrophilic Fe(O) center and a contribution of quantum mechanical tunneling to the reaction.Phenylalanine hydroxylase (PheH) is a non-heme iron dependent monooxygenase that catalyzes the hydroxylation of the amino acid phenylalanine to yield tyrosine (Scheme 1) 1 . PheH is found in organisms ranging from bacteria to humans. In mammals, the enzyme is responsible for catabolism of dietary phenylalanine, and mutations in PheH are linked to the disorder phenylketonuria 2 . Among the bacterial enzymes, that from Chromobacterium violaceum (CvPheH) is the most studied3 -6. PheH is a member of the family of aromatic amino acid hydroxylases, along with tyrosine hydroxylase (TyrH) and tryptophan