Analytical ultracentrifugation has been used to analyze the oligomeric structure of the isolated regulatory domain of phenylalanine hydroxylase. The protein exhibits a monomer–dimer equilibrium with a dissociation constant of ∼46 μM; this value is unaffected by the removal of the 24 N-terminal residues or by phosphorylation of Ser16. In contrast, phenylalanine binding (Kd = 8 μM) stabilizes the dimer. These results suggest that dimerization of the regulatory domain of phenylalanine hydroxylase is linked to allosteric activation of the enzyme.

The aromatic amino acid hydroxylases tryptophan hydroxylase and tyrosine hydroxylase are responsible for the initial steps in the formation of serotonin and the catecholamine neurotransmitters, respectively. Both enzymes are nonheme iron-dependent monooxygenases that catalyze the insertion of one atom of molecular oxygen onto the aromatic ring of their amino acid substrates, using a tetrahydropterin as a two electron donor to reduce the second oxygen atom to water. This review discusses the current understanding of the catalytic mechanism of these two enzymes. The reaction occurs as two sequential half reactions: a reaction between the active site iron, oxygen, and the tetrahydropterin to form a reactive FeIVO intermediate and hydroxylation of the amino acid by the FeIVO. The mechanism of formation of the FeIVO is unclear; however, considerable evidence suggests the formation of an FeII-peroxypterin intermediate. The amino acid is hydroxylated by the FeIVO intermediate in an electrophilic aromatic substitution mechanism.

The antischistosomal prodrug oxamniquine is activated by a sulfotransferase (SULT) in the parasitic flatworm Of the three main human schistosome species, only is sensitive to oxamniquine therapy despite the presence of SULT orthologs in and The reason for this species-specific drug action has remained a mystery for decades. Here we present the crystal structures of and SULTs, including SULT in complex with oxamniquine. We also examined the activity of the three enzymes ; surprisingly, all three are active toward oxamniquine, yet we observed differences in catalytic efficiency that implicate kinetics as the determinant for species-specific toxicity. These results provide guidance for designing oxamniquine derivatives to treat infection caused by all species of schistosome to combat emerging resistance to current therapy.

The flavoprotein L-hydroxynicotine oxidase (LHNO) catalyzes an early step in the bacterial catabolism of nicotine. Although, the structure of the enzyme establishes that it is a member of the monoamine oxidase family, LHNO is generally accepted to oxidize a carbon-carbon bond in the pyrrolidine ring of the substrate and has been proposed to catalyze the subsequent tautomerization and hydrolysis of the initial oxidation product to yield 6-hydroxypseudooxynicotine (Kachalova et al. (2011) Proc. Natl. Acad. Sci. USA 108, 4800–4805). Analysis of the product of the enzyme from Arthrobacter nicotinovorans by NMR and continuous-flow mass spectrometry establishes that the enzyme catalyzes the oxidation of the pyrrolidine carbon-nitrogen bond, the expected reaction for a monoamine oxidase, and that hydrolysis of the amine to form 6-hydroxypseudooxynicotine is nonenzymatic. Based on the kcat/Km and kred values for (S)-hydroxynicotine and several analogs, the methyl group contributes only marginally (~0.5 kcal/mol) to transition state stabilization, while the hydroxyl oxygen and pyridyl nitrogen each contribute ~4 kcal/mol. The small effects on activity of mutagenesis of His187, Glu300, or Tyr407 rule out catalytic roles for all three of these active-site residues.

Intron lariats are circular, branched RNAs (bRNAs) produced during pre-mRNA splicing. Their unusual chemical and topological properties arise from branch-point nucleotides harboring vicinal 2′,5′-and 3′,5′-phosphodiester linkages. The 2′,5′-bonds must be hydrolyzed by the RNA debranching enzyme Dbr1 before spliced introns can be degraded or processed into small nucleolar RNA and microRNA derived from intronic RNA. Here, we measure the activity of Dbr1 from Entamoeba histolytica by using a synthetic, dark-quenched bRNA substrate that fluoresces upon hydrolysis. Purified enzyme contains nearly stoichiometric equivalents of Fe and Zn per polypeptide and demonstrates turnover rates of ∼3 s −1. Similar rates are observed when apo-Dbr1 is reconstituted with Fe(II)+Zn(II) under aerobic conditions. Under anaerobic conditions, a rate of ∼4.0 s −1 is observed when apoenzyme is reconstituted with Fe(II). In contrast, apo-Dbr1 reconstituted with Mn(II) or Fe(II) under aerobic conditions is inactive. Diffraction data from crystals of purified enzyme using X-rays tuned to the Fe absorption edge show Fe partitions primarily to the β-pocket and Zn to the α-pocket. Structures of the catalytic mutant H91A in complex with 7-mer and 16-mer synthetic bRNAs reveal bona fide RNA branchpoints in the Dbr1 active site. A bridging hydroxide is in optimal position for nucleophilic attack of the scissile phosphate. The results clarify uncertainties regarding structure/function relationships in Dbr1 enzymes, and the fluorogenic probe permits high-throughput screening for inhibitors that may hold promise as treatments for retroviral infections and neurodegenerative disease.RNA debranching | intron lariat | enzyme kinetics | X-ray crystallography | Dbr1 T he enzymatic processing of diverse RNA molecules requires selective recognition of their unique physicochemical properties. The sequential trans-esterification reactions catalyzed by the spliceosome yield mature messenger RNA (mRNA) and excised intron lariats (1, 2), the latter of which contain internal branchpoint adenosine nucleotides harboring vicinal 2′,5′-and 3′,5′-phosphodiester linkages (3). Mature mRNA transcripts are exported to the cytosol for protein synthesis, but lariat introns must be linearized before they can be turned over or processed into the subset of small nucleolar RNAs and microRNAs that are derived from intronic RNA (4, 5). The lariat forms when the 2′-hydroxyl group of an adenosine nucleotide near the 3′-end of the intron acts as the nucleophile to attack the 5′-splice site, producing 5′-exon-3′-OH and intron lariat/ 3′-exon intermediates. The 3′-hydroxyl group of the 5′-exon-3′-OH intermediate subsequently acts as the nucleophile to attack the 3′-splice site, resulting in intron excision and exon ligation (6, 7) (Fig. 1A). The resulting vicinal 2′,5′-and 3′,5′-phosphodiester linkages confer unique topological and chemical features to the branchpoint and flanking nucleotides, and these lariats persist in yeast cells lacking active Dbr1 (RNA lariat debranching enzyme) ...