Biochemistry 45:10407C10411. compared to additional reported amidohydrolase 2 family decarboxylases. Moreover, PicC was found to form a monophyletic group in the phylogenetic tree constructed using PicC and related proteins. Further, the genetic deletion and complementation results shown that was essential for PA degradation. The PicC was Zn2+-dependent nonoxidative decarboxylase that can specifically catalyze the irreversible decarboxylation of 3,6DHPA to 2,5-dihydroxypyridine. The and (27), (28), (29), (30), (31), (32), and (33). The metabolic pathway of PA in microorganisms has been partially elucidated in earlier studies (15, 28, 32) (Fig. 1). In additional studies, the crude enzyme facilitating the conversion of PA to 6-hydroxypicolinic acid (6HPA) has been preliminarily purified in DSM 20665 and an unidentified Gram-negative bacterium (designated the UGN strain) (30, 34). However, the practical genes or enzymes involved in PA degradation have not been cloned or characterized yet. Open in a separate windowpane FIG 1 Proposed PA degradation pathway in JQ135. Dotted arrows show the proposed methods. The 3,6DHPA and 2,5-DHP are demonstrated in blue. TCA, tricarboxylic acid cycle. In our earlier work, we shown that strain JQ135 utilizes PA as the sole carbon and nitrogen resource and as an energy source and that 6-hydroxypicolinic acid (6HPA) was the 1st intermediate of PA (35). Further studies showed the gene was essential for PA catabolism (36). In the present study, we statement the fully characterized intermediate compound, 3,6-dihydroxypicolinic acid (3,6DHPA) (Fig. 1). Further, a novel nonoxidative 3,6-dihydroxypicolinic acid decarboxylase gene (strain JQ135, and the related product was characterized. RESULTS Transposon mutant and recognition of the intermediate 3,6DHPA. A library of JQ135 mutants incapable of 6HPA utilization was constructed by random transposon mutagenesis. More than 30 mutants that could not Rabbit Polyclonal to MMP15 (Cleaved-Tyr132) grow on 6HPA-containing medium were selected from approximately 10,000 clones and their ability to convert 6HPA was examined. High-performance liquid chromatography (HPLC) results showed that one mutant (designated Mut-H4) could convert 6HPA into a fresh intermediate with no further degradation (Fig. 2). After liquid chromatography/time of flight-mass spectrometry (LC/TOF-MS) analysis, it was found that the molecular ion maximum ([M+H]+) of this fresh intermediate was 156.0295 (ion formula, C6H6NO4+; determined molecular excess weight, 156.0297 with ?3.2?ppm error), indicating that one oxygen atom was added to 6HPA (C6H5NO3). According to the Carisoprodol previously expected PA degradation pathway, the intermediate is most likely to be 3,6DHPA (15, 31, 34). In the present study, 3,6DHPA was chemically synthesized and characterized by UV-visible spectroscopy (UV-VIS), LC/TOF-MS, 1H nuclear magnetic resonance (NMR), and 13C NMR spectroscopies (observe Fig. Carisoprodol S1 and S2 in the supplemental material) and HPLC analysis showed the retention time of the new intermediate was identical to that of the synthetic sample of 3,6DHPA (Fig. 2). Therefore, this intermediate compound was identified as 3,6DHPA. Open in a separate windowpane FIG 2 HPLC and LC/TOF-MS profiles of the conversion of 6HPA by mutant Mut-H4. (A and C) The authentic samples of 6HPA and 3,6DHPA, respectively. (B) Conversion of 6HPA into 3,6DHPA by mutant Mut-H4. The detection wavelength was arranged at 310?nm. (D) LC/TOF-MS spectra of 3,6DHPA produced in panel B. Screening of the 3,6DHPA decarboxylase gene. The transposon insertion site of mutant Mut-H4 was recognized using the genome walking method (37). The insertion site of the transposon was located in gene (genome position 3298929). Gene was a 972-bp size open reading framework (ORF) starting with GTG. exhibited the highest sequence similarity to several nonoxidative decarboxylases such as (designated gene in PA degradation in JQ135. To confirm whether is involved in PA degradation, was constructed. The mutant JQ135lost the ability to grow on Carisoprodol PA, 6HPA, or 3,6DHPA. The complemented strain, JQ135was essential for the degradation Carisoprodol of PA in JQ135. encodes Carisoprodol a 3,6DHPA.