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The Novel Monocomponent FAD-Dependent Monooxygenase Hpam Catalyzes

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The Novel Monocomponent FAD-Dependent Monooxygenase Hpam Catalyzes bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 The Novel Monocomponent FAD-dependent Monooxygenase HpaM Catalyzes 2 the 2-Decarboxylative Hydroxylation of 5-Hydroxypicolinic Acid in Alcaligenes 3 faecalis JQ135 4 5 Jiguo Qiu,a Bin Liu,a Lingling Zhao,a Yanting Zhang,a Dan Cheng,b Xin Yan, a ∗ 6 Jiandong Jiang,a Qing Hong,a and Jian He a 7 8 a Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of 9 Life Sciences, Nanjing Agricultural University, Nanjing, 210095, China 10 b Laboratory Centre of Life Science, College of Life Sciences, Nanjing Agricultural University, 11 Nanjing, 210095, China 12 13 ∗Address correspondence to Jian He 14 Email: [email protected]; Tel: (86)-25-84396685, Fax: (86)-25-84396314 15 16 Runing title: 5-Hydroxypicolinic Acid 2-Monooxygenase HpaM 17 18 Key words: Alcaligenes faecalis JQ135, 5-hydroxypicolinic acid, biodegradation, 19 5-hydroxypicolinic acid 2-monooxygenase, 2-decarboxylative hydroxylation 20 21 1 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 22 Abstract 23 5-hydroxypicolinic acid (5HPA) is a natural pyridine derivative that can be 24 microbially degraded. However, the physiological, biochemical, and genetic 25 foundation of the microbial catabolism of 5HPA remains unknown. In this study, a 26 gene cluster hpa (which is involved in degradation of 5HPA in Alcaligenes faecalis 27 JQ135) was cloned and HpaM was identified as a novel monocomponent 28 FAD-dependent monooxygenase. HpaM shared a sequence only 31% similarity with 29 the most related protein 6-hydroxynicotinate 3-monooxygenase (NicC) of 30 Pseudomonas putida KT2440. hpaM was heterologously expressed in E. coli 31 BL21(DE3), and the recombinant HpaM was purified via Ni-affinity chromatography. 32 HpaM catalyzed the 2-decarboxylative hydroxylation of 5-HPA, thus generating 33 2,5-dihydroxypyridine (2,5-DPH). Monooxygenase activity was only detected in the 34 presence of FAD and NADH, but not of FMN and NADPH. The apparent Km values 35 of HpaM toward 5HPA and NADH were 45.4 μΜ and 37.8 μΜ, respectively. Results 36 of gene deletion and complementation showed that hpaM was essential for 5HPA 37 degradation in Alcaligenes faecalis JQ135. 38 39 2 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 40 Importance 41 Pyridine derivatives are ubiquitous in nature and important chemical materials 42 that are currently widely used in agriculture, pharmaceutical, and chemical industries. 43 Thus, the microbial degradation and transformation mechanisms of pyridine 44 derivatives received considerable attention. Decarboxylative hydroxylation was an 45 important degradation process in pyridine derivatives, and previously reported 46 decarboxylative hydroxylations happened in the C3 of the pyridine ring. In this study, 47 we cloned the gene cluster hpa, which is responsible for 5HPA degradation in 48 Alcaligenes faecalis JQ135, thus identifying a novel monocomponent FAD-dependent 49 monooxygenase HpaM. Unlike 3-decarboxylative monooxygenases, HpaM catalyzed 50 decarboxylative hydroxylation in the C2 of the pyridine ring in 5-hydroxypicolinic 51 acid. These findings deepen our understanding of the molecular mechanism of 52 microbial degradation of pyridine derivatives. Furthermore, HpaM offers potential for 53 applications to transform useful pyridine derivatives. 54 55 3 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 56 Introduction 57 Pyridine derivatives are common natural products, as well as important artificial 58 compounds that are widely used in agriculture, pharmaceutical, and chemical 59 industries as solvents, dyes, pharmaceuticals, herbicides, and pesticides (1-3). 60 However, the increasing use of pyridine derivatives causes large amounts entering the 61 environment, thus leading to severe environmental problems (4, 5). Therefore, the 62 biodegradation or detoxication of pyridine derivatives, and their transformation to 63 useful products are of significant interest. 64 Pyridine derivatives could either be degraded or transformed by a variety of 65 bacteria, and the degradation processes are typically initialed via hydroxylation (6, 7). 66 Nicotinic acid (NA, 3-pyridinecarboxylic acid) and nicotine often served as models 67 for explore the catabolic mechanisms of pyridine derivatives (8-11). NA was initially 68 hydroxylated at the C2 of the pyridine ring via NA monooxygenase (NicAB), thus 69 producing 6-hydroxynicotinic acid (6HNA) (8). 6HNA was further decarboxylatively 70 hydroxylated at the C3 of the pyridine ring via 6HNA monooxygenase (NicC) 71 yielding 2,5-dihydroxypyridine (2,5-DHP), which was then subjected to ring-cleavage. 72 Nicotine could be degraded via both the pyridine and pyrrolidine pathways, and both 73 pathways include two hydroxylation steps. In the pyrrolidine pathway, the 74 intermediate 3-succinoylpyridine (SP) was hydroxylated at the C2 of the pyridine ring 75 via SP monoxygenase (Spm), thus generating 6-hydroxy-3-succinoylpyridine (HSP), 76 which was further 3-decarboxylatively hydroxylated to 2,5-DHP via HSP 77 monoxygenase (HspB). In the pyridine pathway, nicotine was hydroxylated at the C6 78 of the pyridine ring via nicotine hydroxylase (NDH) to 6-hydroxynicotine, while the 79 downstream intermediate 2,6-dihydroxypyridine was 3-hydroxylated to 80 2,3,6-trihydroxypyridine via 2,6-dihydroxypyridine 3-monoxygenase (DHPH) (10, 4 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 81 12). NicAB and Spm are multicomponent molybdenum-containing monooxygenases, 82 while the NicC and HspB are monocomponent flavin-dependent monooxygenases, 83 catalyzing the 3-decarboxylatively hydroxylation. However, gene coding of 84 monooxygenase catalyzing the 2-decarboxylatively hydroxylation of pyridine 85 derivatives has not been reported to date. 86 5-Hydroxypicolinic acid (5HPA) is a isomer of 6HNA and a natural pyridine 87 derivative produced by bacteria (such as Nocardia sp.) or plants (such as Gynura 88 divaricata) (13, 14). The degradation of 5HPA has only been reported in Pusillimonas 89 sp. 5HP (15). A 5-hydroxypicolinate 2-monooxygenase (catalyzing the 90 2-decarboxylative hydroxylation of 5-HPA to 2,5-DHP) was partially purified from 91 strain 5HP. However, both the amino acid sequence of the 5-hydroxypicolinate 92 2-monooxygenase, and the genetic foundation of 5HPA degradation remain unknown. 93 In this study, the gene cluster hpa involved in 5HPA degradation was cloned, and 94 a 5-hydroxypicolinic acid 2-monooxygenase HpaM was identified from Alcaligenes 95 faecalis JQ135 (Fig. 1A, B). HpaM is FAD and NADH-dependent, and catalyzes the 96 2-decarboxylative hydroxylation of the pyridine-ring in 5HPA to produce 2,5-DHP. 97 98 Results 99 Degradation of 5HPA by strain A. faecalis JQ135. 100 A. faecalis JQ135 was formerly identified as a picolinic acid (PA)-degrading 101 bacterium (16). 5HPA is a 5-hydroxylated derivate of PA that was tested for 102 degradation and utilization by the strain in a carbon and nitrogen-absent MSM. The 103 results showed that the strain A. faecalis JQ135 could completely degrade 1 mM 5HPA 104 within 36 h, and correspondingly, the OD600 of the culture increased from 0.2 to 0.5 105 ± 0.1 (Fig. 2). These results indicated that strain A. faecalis JQ135 could degrade and 5 / 26 bioRxiv preprint doi: https://doi.org/10.1101/171595; this version posted August 14, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 106 utilize 5HPA as sole source of carbon and nitrogen for growth. In addition, attempts to 107 detect metabolic intermediates of 5HPA within the culture failed. This might because 108 little or no intermediates were excreted from strain JQ135 cells during degradation of 109 5HPA. 110 111 Transposon mutagenesis and cloning of a gene cluster involved in 5HPA 112 degradation 113 To clone the genes involved in 5HPA degradation, a transposon mutagenesis 114 library of A. faecalis JQ135 was constructed. One mutant (Z10) could not grow in 115 MSM agar containing 1 mM 5HPA and was screened from approximately 5000 116 mutants. When inoculated into liquid MSM, containing 1 mM 5HPA, mutant Z10 117 could not degrade 5HPA (Fig. 2). Furthermore, the genome of A. faecalis JQ135 was 118 determined by the PacBio system. The complete genome of the strain contained one 119 circular chromosome (4,078,346 bp) and no plasmid could be found. A total of 3,723 120 ORFs were predicted. The insertion position of the transposon, determined via the 121 DNA walking method (17), was located in gene AFA_18575 (genome position 122 4,070,825). AFA_18575 is 1,218 bp in length with a G+C content of 56.08%. The 123 deduced protein was searched against the NCBI database 124 (http://blast.ncbi.nlm.nih.gov/), using the BLASTP program (Table 1). The results 125 showed that the proteins that were related the most were flavin-containing 126 monooxygenases, such as 6-hydroxynicotinate 3-monooxygenase (NicC, sequence ID: 127 Q88FY2) from Pseudomonas putida KT2440 (identity of 31%) (8), and salicylate 128 1-monooxygenase (SalM or NahG, sequence ID: P23262) from P.
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