Catabolism of 3-Hydroxypyridine by Ensifer Adhaerens HP1: a Novel Four-Component

bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.898148; this version posted January 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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 Title: 2 Catabolism of 3-hydroxypyridine by Ensifer adhaerens HP1: a novel four-component 3 gene encoding 3-hydroxypyridine dehydrogenase HpdA catalyzes the first step of 4 biodegradation 5 6 Running title: 7 Microbial 3-hydroxypyridine degradation 8 9 Authors: 10 Haixia Wang a, Xiaoyu Wang a, Hao Ren a, Xuejun Wang a, Zhenmei Lu a# 11 12 a MOE Laboratory of Biosystem Homeostasis and Protection, College of Life Sciences, 13 Zhejiang University, Hangzhou, China 14 15 #Address correspondence to Zhenmei Lu, [email protected] 16 17 18 19 20 21 22 23 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.898148; this version posted January 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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. 24 Abstract 25 3-Hydroxypyridine (3HP) is an important natural pyridine derivative. Ensifer 26 adhaerens HP1 can utilize 3HP as the sole source of carbon, nitrogen and energy to 27 grow. However, the genes responsible for the degradation of 3HP remain unknown. In 28 this study, we predicted that a gene cluster, designated 3hpd, may be responsible for the 29 degradation of 3HP. The initial hydroxylation of 3HP is catalyzed by a four-component 30 dehydrogenase (HpdA1A2A3A4), leading to the formation of 2,5-dihydroxypyridine 31 (2,5-DHP) in E. adhaerens HP1. In addition, the SRPBCC component in HpdA existed 32 as a separate subunit, which is different from other SRPBCC-containing 33 molybdohydroxylases acting on N-heterocyclic aromatic compounds. Our findings 34 provide a better understanding of the microbial degradation of pyridine derivatives in 35 nature. Additionally, research on the origin of the discovered four-component 36 dehydrogenase with a separate SRPBCC domain may be of great significance. 37 38 Importance 39 3-Hydroxypyridine is an important building block for synthesizing drugs, herbicides 40 and antibiotics. Although the microbial degradation of 3-hydroxypyridine has been 41 studied for many years, the molecular mechanisms remain unclear. Here, we show that 42 3hpd is responsible for the catabolism of 3-hydroxypyridine. The 3hpd gene cluster was 43 found to be widespread in Actinobacteria, Rubrobacteria, Thermoleophilia, and Alpha-, 44 Beta-, and Gammaproteobacteria, and the genetic organization of the 3hpd gene 45 clusters in these bacteria showed high diversity. Our findings provide new insight into 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.898148; this version posted January 9, 2020. 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. 46 the catabolism of 3-hydroxypyridine in bacteria. 47 48 Keywords 49 3-hydroxypyridine, Ensifer adhaerens HP1, 3-hydroxypyridine catabolism, 3- 50 hydroxypyridine dehydrogenase, 3hpd 51 52 Introduction 53 The pyridine ring is a major constituent of natural compounds such as plant alkaloids, 54 coenzymes and antibiotics. 3-Hydroxypyridine (3HP), a useful and valuable pyridine 55 derivative, is a monohydroxypyridine in which the hydrogen at position 3 of the 56 pyridine has been replaced by a hydroxyl group. 3HP has been detected as a thermal 57 degradation product in the smoke from burning Salvia divinorum leaves and as a 58 significant constituent of tobacco smoke[1, 2]. Many bioactive compounds contain 3HP 59 as an important structural unit, and 3HP is widely used as a building block to synthesize 60 drugs, herbicides, insecticides and antibiotics[3-5]. Large amounts of 3HP are 61 synthesized each year, and there are many synthetic methods for 3HP, such as 62 ruthenium-catalyzed ring-closing olefin metathesis[6]. The widespread use of 3HP has 63 made its release to the environment inevitable, which may have serious implications 64 for human health. While 3HP can be eliminated by physical and chemical methods, 65 microbial biodegradation has been considered one of the most economical and effective 66 approaches to remediating 3HP pollution. 67 Catabolism of pyridine, particularly the initial steps of the hydroxylation of 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.898148; this version posted January 9, 2020. 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. 68 monohydroxylated pyridines such as 2-hydroxypyridine (2HP), 3HP and 4- 69 hydroxypyridine (4HP), has received much attention. In particular, numerous bacteria 70 have been reported to use 2HP as the sole carbon and energy source to grow. Many 71 intermediates have been identified, and metabolic pathways have been proposed for 72 2HP biodegradation. One pathway of 2HP biodegradation involves the formation of 73 2,5-dihydroxypyridine (2,5-DHP), which then proceeds through the maleamate 74 pathway. The other pathway, involving the formation of 2,3,6-trixydroxypyridine first, 75 produces a blue pigment (nicotine blue) in the medium. Rhodococcus rhodochrous 76 PY11 was reported to use 2HP as the sole source of carbon and energy through the 77 nicotine blue-production pathway. A gene cluster (hpo) has been characterized as being 78 responsible for the catabolism of 2HP in strain PY11, and the initial hydroxylation of 79 2HP is catalyzed by a four-component dioxygenase (HpoBCDF)[7]. Burkholderia sp. 80 MAK1 was also reported to degrade 2HP, but through the maleamate pathway. A gene 81 cluster (hpd) was responsible for the degradation of 2HP in strain MAK1, and the 2- 82 hydroxypyridine 5-monooxygenase is a soluble di-iron monooxygenase (SDIMO) 83 encoded by a five-gene cluster hpdABCED[8]. Several strains have been reported to 84 partially or completely degrade 3HP. Achromobacter sp. (G2 and 2L)[9], Pusillimonas 85 sp. 5HP[10] and Agrobacterium sp. DW-1[11] could use 3HP as a carbon and nitrogen 86 source to grow. Nocardia Z1 was reported to slowly oxidize 3HP to pyridine-2,3-diol 87 and pyridine-3,4-diol[12], while Achromobacter 7N could convert 3HP to only 2,5-DHP 88 and could hardly further metabolize the diol[9]. 89 Moreover, microbial degradation of 3HP, which has been proposed for several different 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.898148; this version posted January 9, 2020. 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. 90 strains, was thought to proceed via the maleamate pathway[9, 12-14], 91 3HP →2,5-DHP → formate + maleamate → NH3 + maleate ↔ fumarate, 92 and was recently confirmed in strain DW-1[11]. However, the genes and enzymes 93 responsible for 3HP biodegradation have seldom been reported. Pyridine-2,5-diol 94 dioxygenase has been partially purified and characterized in only strains G2 and 2L[9]. 95 The pyridine-2,5-diol dioxygenase in these two strains required Fe2+ to restore full 96 activity after purification, and the hydroxylases of strains G2 and 2L showed clear 97 specificity because they produced only the para-substituted 2,5-DHP from 3HP. No 98 enzymes responsible for the initial hydroxylation step of 3HP leading to the formation 99 of 2,5-DHP have been reported to date. 100 In this study, the bacterial strain Ensifer adhaerens HP1 was isolated from soil and 101 showed effective degradation and utilization of 3HP. We report the isolation and 102 characterization of the 3HP catabolic pathway in E. adhaerens HP1. A gene cluster 103 (3hpd) encoding the putative proteins required for 3HP biodegradation in this bacterium 104 was discovered and characterized. The results of bioinformatics analysis, gene 105 knockout and complementation of hpdA, and heterologous expression of HpdA suggest 106 that multicomponent HpdA is involved in the transformation of 3HP to 2,5-DHP. 107 108 Results and discussion 109 Isolation of a bacterium capable of degrading 3HP 110 A bacterium was isolated from enrichments of soil with 3HP and was selected for 111 detailed study. This strain could utilize 3HP as the sole source of carbon, nitrogen and 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.898148; this version posted January 9, 2020. 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. 112 energy to grow (Figure 1) and was designated HP1. The 16S rRNA sequence of strain 113 HP1 exhibited the highest similarity (99%) to E. adhaerens strain Casida A (accession 114 number: CP015882.1). Therefore, strain HP1 was identified as E. adhaerens HP1. 115 During the growth of strain HP1 on 3HP-containing media, a green pigment 116 accumulated and gradually turned dark brown. This phenomenon has been reported in 117 strains Pusillimonas sp. 5HP[10], P. putida S16[15], and Agrobacterium sp. S33[16]. All 118 these strains converted the corresponding substrate to 2,5-DHP, and this color change 119 indicated the formation of 2,5-DHP. 2,5-DHP was detected in the supernatant of strain 120 HP1 by LC-MS analysis (Figure S1), suggesting that the degradation of 3HP in strain 121 HP1 occurs through the maleamate pathway (Figure 2B). Moreover, strain HP1 utilized 122 only 3HP but not the other two hydroxypyridine isomers, 2HP and 4HP (data not 123 shown). 124 125 A putative 3HP metabolism gene cluster is present in the genome of strain HP1 126 After performing a BLAST analysis against the genome of strain HP1 using previously 127 known metabolic genes involved in the maleamate pathway, we found a putative 128 metabolic gene cluster, hpdDBCE, in the genome of strain HP1 (Figure 2A).

Load more