133331-79-0Relevant articles and documents
A Single-Site Mutation at Ser146 Expands the Reactivity of the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase
Dhammaraj, Taweesak,Pinthong, Chatchadaporn,Visitsatthawong, Surawit,Tongsook, Chanakan,Surawatanawong, Panida,Chaiyen, Pimchai
, p. 2889 - 2896 (2016)
The oxygenase component (C2) of p-hydroxyphenylacetate (4-HPA) 3-hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of various phenolic acids. In this report, we found that substitution of a residue close to the phenolic group binding site to yield the S146A variant resulted in an enzyme that is more effective than the wild-type in catalyzing the hydroxylation of 4-aminophenylacetate (4-APA). Product yields for both wild-type and S146A enzymes are better at lower pH values. Multiple turnover reactions of the wild-type and S146A enzymes indicate that both enzymes first hydroxylate 3-APA to give 3-hydroxy-4-aminophenylacetate (3-OH-4-APA), which is further hydroxylated to give 3,5-dihydroxy-4-aminophenylacetate, similar to the reaction of C2 with 4-HPA. Stopped-flow experiments showed that 4-APA can only bind to the wild-type enzyme at pH 6.0 and not at pH 9.0, while it can bind to S146A under both pH conditions. Rapid-quench flow results indicate that the wild-type enzyme has low reactivity toward 4-APA hydroxylation, with a hydroxylation rate constant (kOH) for 4-APA of 0.028 s-1 compared to 17 s-1 for 4-HPA, the native substrate. In contrast, for S146A, the hydroxylation rate constants for both substrates are very similar (2.6 s-1 for 4-HPA versus 2.5 s-1 for 4-APA). These data indicate that Ser146 is a key catalytic residue involved in optimizing C2 reactivity toward a phenolic compound. Removing this hydroxyl group expands C2 activity toward a non-natural aniline substrate. This understanding should be helpful for future rational engineering of other two-component flavin-dependent monooxygenases that have this conserved Ser residue.
Reduction of the nitro group to amine by hydroiodic acid to synthesize o-aminophenol derivatives as putative degradative markers of neuromelanin
Wakamatsu, Kazumasa,Tanaka, Hitomi,Tabuchi, Keisuke,Ojika, Makoto,Zucca, Fabio A.,Zecca, Luigi,Ito, Shosuke
, p. 8039 - 8050 (2014/07/08)
Neuromelanin (NM) is produced in dopaminergic neurons of the substantia nigra (SN) and in noradrenergic neurons of the locus coeruleus (LC). The synthesis of NM in those neurons is a component of brain aging and there is the evidence that this pigment can be involved in the pathogenesis of neurodegenerative diseases such as Parkinson's disease. NM is believed to derive from the oxidative polymerization of dopamine (DA) or norepinephrine (NE) with the participation of cysteine, dolichols and proteins. However, there are still unknown aspects in the chemical structure of NM from SN (SN-NM) and LC (LC-NM). In this study, we designed a new method to synthesize o-aminophenol compounds as putative degradation products of catecholamines and their metabolites which may be incorporated into NM. Those compounds are aminohydroxyphenylethylamine (AHPEA) isomers, aminohydroxyphenylacetic acid (AHPAA) isomers and aminohydroxyethylbenzene (AHEB) isomers, which are expected to arise from DA or NE, 3,4-dihydroxyphenylacetic acid (DOPAC) or 3,4-dihydroxyphenylmandelic acid (DOMA) and 3,4-dihydroxyphenylethanol (DOPE) or 3,4- dihydroxyphenylethyleneglycol (DOPEG), respectively. These o-aminophenol compounds were synthesized by the nitration of phenol derivatives followed by reduction with hydroiodic acid (HI), and they could be identified by HPLC in HI hydrolysates of SN-NM and LC-NM. This degradative approach by HI hydrolysis allows the identification of catecholic precursors unique to SN-NM and LC-NM, which are present in catecholaminergic neurons.
