title: Two unexpected promiscuous activities of the iron-sulfur protein IspH in production of isoprene and isoamylene.
abstract: BACKGROUND
  Bacillus species, possessing the methylerythritol phosphate (MEP) pathway for the synthesis of isoprenoid feedstock, are the highest producers of isoprene among bacteria; however, the enzyme responsible for isoprene synthesis has not been identified. The iron-sulfur protein IspH is the final enzyme of the MEP pathway and catalyses the reductive dehydration of (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMBPP) to form isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP). In this study, we demonstrated two unexpected promiscuous activities of IspH from alkaliphilic Bacillus sp. N16-5, which can produce high levels of isoprene.
  
  
  RESULTS
  Bacillus sp. N16-5 IspH could catalyse the formation of isoprene from HMBPP and the conversion of DMAPP into a mixture of 2-methyl-2-butene and 3-methyl-1-butene. Both reactions require an electron transfer system, such as that used for HMBPP dehydration. Isoprene and isoamylene synthesis in Bacillus sp. N16-5 was investigated and the reaction system was reconstituted in vitro, including IspH, ferredoxin and ferredoxin-NADP(+)-reductase proteins and NADPH. The roles of specific IspH protein residues were also investigated by site-directed mutagenesis experiments; two variants (H131N and E133Q) were found to have lost the HMBPP reductase activity but could still catalyse the formation of isoprene. Overexpression of IspH H131N in Bacillus sp. N16-5 resulted in a twofold enhancement of isoprene production, and the yield of isoprene from the strain expressing E133Q was increased 300% compared with the wild-type strain.
  
  
  CONCLUSIONS
  IspH from Bacillus sp. N16-5 is a promiscuous enzyme that can catalyse formation of isoprene and isoamylene. This enzyme, especially the H131N and E133Q variants, could be used for the production of isoprene from HMBPP.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.|University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China.|College of Medicine, Anhui University of Science and Technology, Huainan, People's Republic of China.
authors.forename: Deyong
authors.initials: D
authors.lastname: Ge
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.
authors.forename: Yanfen
authors.initials: Y
authors.lastname: Xue
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China. mayanhe@im.ac.cn.
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
mesh_terms: D001407:Bacillus / Q000254:growth & development / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002070:Butadienes; D004591:Electrophoresis, Polyacrylamide Gel; D005288:Ferredoxins / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D045782:Hemiterpenes / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D007536:Isomerism; D016297:Mutagenesis, Site-Directed; D010755:Organophosphates / Q000737:chemistry / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010420:Pentanes; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: C467061:4-hydroxy-3-methyl-2-butenyl diphosphate; D001426:Bacterial Proteins; D002070:Butadienes; D005288:Ferredoxins; D045782:Hemiterpenes; D007506:Iron-Sulfur Proteins; D010755:Organophosphates; D009943:Organophosphorus Compounds; D010420:Pentanes; D011994:Recombinant Proteins; C005059:isoprene; C043060:3,3-dimethylallyl pyrophosphate; D010088:Oxidoreductases
keywords: (E)-4-Hydroxy-3-methyl-2-butenyl diphosphate (HMBPP); 2-Methyl-2-butene; 3-Methyl-1-butene; Bacillus; Dimethylallyl diphosphate (DMAPP); Isoprene; Isoprene synthase; IspH; IspS