We have characterized the gene encoding fatty acid alpha-hydroxylase, a cytochrome P450 (P450) enzyme, from Sphingomonas paucimobilis. A database homology search indicated that the deduced amino acid sequence of this gene product was 44% identical to that of the ybdT gene product that is a 48 kDa protein of unknown function from Bacillus subtilis. In this study, we cloned the ybdT gene and characterized this gene product using a recombinant enzyme to clarify function of the ybdT gene product. The carbon monoxide difference spectrum of the recombinant enzyme showed the characteristic one of P450. In the presence of H2O2, the recombinant ybdT gene product hydroxylated myristic acid to produce beta-hydroxymyristic acid and alpha-hydroxymyristic acid which were determined by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry. The amount of these products increased with increasing reaction period and amount of H2O2 in the reaction mixture. The amount of beta-hydroxyl product was slightly higher than that of alpha-hydroxyl product at all times during the reaction. However, no reaction products were detected at any time or at any concentration of H2O2 when heat-inactivated enzyme was used. HPLC analysis with a chiral column showed that the beta-hydroxyl product was nearly enantiomerically pure R-form. These results suggest that this P450 enzyme is involved in a novel biosynthesis of beta-hydroxy fatty acid.

Fatty acid alpha-hydroxylase from Sphingomonas paucimobilis is an unusual cytochrome P450 enzyme that hydroxylates the alpha-carbon of fatty acids in the presence of H2O2. Herein, we describe our investigation concerning the utilization of various substrates and the optical configuration of the alpha-hydroxyl product using a recombinant form of this enzyme. This enzyme can metabolize saturated fatty acids with carbon chain lengths of more than 10. The Km value for pentadecanoic acid (C15) was the smallest among the saturated fatty acids tested (C10-C18) and that for myristic acid (C14) showed similar enzyme kinetics to those seen for C15. As shorter or longer carbon chain lengths were used, Km values increased. The turnover numbers for fatty acids with carbon chain lengths of more than 11 were of the same order of magnitude (10(3) min(-1)), but the turnover number for undecanoic acid (C11) was less. Dicarboxylic fatty acids and methyl myristate were not metabolized, but monomethyl hexadecanedioate and omega-hydroxypalmitic acid were metabolized, though with lower turnover values. Arachidonic acid was a good substrate, comparable to C14 or C15. The metabolite of arachidonic acid was only alpha-hydroxyarachidonic acid. Alkanes, fatty alcohols, and fatty aldehydes were not utilized as substrates. Analysis of the optical configurations of the alpha-hydroxylated products demonstrated that the products were S-enantiomers (more than 98% enantiomerically pure). These results suggested that this P450 enzyme is strictly responsible for fatty acids and catalyzes highly stereo- and regioselective hydroxylation, where structure of omega-carbon and carboxyl carbon as well as carbon chain length of fatty acids are important for substrate-enzyme interaction.

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Introduction: Several antigen tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed worldwide, but their clinical utility has not been well established. In this study, we evaluated the analytical and clinical performance of QuickNavi™-COVID19 Ag, a newly developed antigen test in Japan. Methods: This prospective observational study was conducted at a PCR center between October 7 and December 5, 2020. The included patients were referred from a local public health center and 89 primary care facilities. We simultaneously obtained two nasopharyngeal samples with flocked swabs; one was used for the antigen test and the other for real-time reverse transcription PCR (RT-PCR). Using the results of real-time RT-PCR as a reference, the performance of the antigen test was evaluated. Results: A total of 1186 patients were included in this study, and the real-time RT-PCR detected SARS-CoV-2 in 105 (8.9%). Of these 105 patients, 33 (31.4%) were asymptomatic. The antigen test provided a 98.8% (95% confidence interval [CI]: 98.0%e99.4%) concordance rate with real-time RT-PCR, along with a sensitivity of 86.7% (95% CI: 78.6%e92.5%) and a specificity of 100% (95% CI: 99.7%e100%). False-negatives were observed in 14 patients, 8 of whom were asymptomatic and had a low viral load (cycle threshold (Ct) > 30). In symptomatic patients, the sensitivity was 91.7% (95% CI: 82.7%e96.9%). Conclusion: QuickNavi™-COVID19 Ag showed high specificity and sufficient sensitivity for the detection of SARS-CoV-2. This test is a promising potential diagnostic modality especially in symptomatic patients.

The clinical utility of antigen test using anterior nasal samples has not been well evaluated. We conducted a prospective study in a drive-through testing site located at a PCR center to evaluate the diagnostic performance of the antigen test QuickNavi-COVID19 Ag using anterior nasal samples and to compare the degrees of coughs or sneezes induction and the severity of pain between anterior nasal collection and nasopharyngeal collection. The study included a total of 862 participants, of which 91.6% were symptomatic. The median duration from symptom onset to sample collection was 2.0 days. Fifty-one participants tested positive for severe acute respiratory syndrome coronavirus 2 on reverse transcription PCR (RT-PCR) with nasopharyngeal samples, and all of them were symptomatic. In comparison to the findings of RT-PCR, the antigen test using anterior nasal samples showed 72.5% sensitivity (95% confidence interval [CI] 58.3–84.1%) and 100% specificity (95% CI 99.3–100%). Anterior nasal collection was associated with a significantly lower degree of coughs or sneezes induction and the severity of pain in comparison to nasopharyngeal collection (p < 0.001). The antigen test using anterior nasal samples showed moderate sensitivity in symptomatic patients who were at the early stages of the disease course but was less painful and induced fewer coughs or sneezes.