Abstract
Pseudomonas sp. NCIM 5235 is a caffeine-degrading bacterial strain that metabolizes caffeine by sequential demethylation using methylxanthine demethylases. These enzymes belong to the class of two-component Rieske oxygenases and require an oxidoreductase, NdmD, for efficient catalysis. NdmD in Pseudomonas sp. has a unique domain fusion in its N-terminal that is not observed in any other Rieske oxygenase reductases reported so far. In this report, a ~ 1.7 kb ndmD gene from the gDNA of Pseudomonas sp. has been isolated and has been cloned in a pET28a expression vector. Soluble NdmD was over-expressed in Escherichia coli BL21 cells and purified by Ni2+ NTA chromatography. Monomeric molecular mass of the protein was found to be ~ 65 kDa and optimal activity was observed at 35 °C and pH 8.0. It showed broad substrate specificity with highest Kcat/km of 490.8 ± 17.7 towards cytochrome c. To determine the role of N-terminal Rieske domain in its reductase activity, two deletion constructs Δ114NdmD and Δ250NdmD were made. Cytochrome c reductase (ccr) activity of the NdmD constructs and demethylase activity of NdmA in the presence of NdmD constructs showed that there is no significant difference in the catalytic activity of NdmD upon deletion of its N-terminal Rieske domain. However, there might be some functional and evolutionary significance for the fusion of Rieske domain to NdmD and we hypothesize that this domain fusion is an intermediate phase of evolution towards the development of a more efficient enzyme system for xenobiotic degradation.
Similar content being viewed by others
References
Abu-Soud HM, Yoho LL, Stuehr DJ (1994) Calmodulin controls neuronal nitric-oxide synthase by a dual mechanism. Activation of intra- and interdomain electron transfer. J Biol Chem 269:32047–32050
Ashikawa Y, Fujimoto Z, Noguchi H, Habe H, Omori T, Yamane H, Nojiri H (2006) Electron transfer complex formation between oxygenase and ferredoxin components in Rieske nonheme iron oxygenase system. structure 14:1779–1789. https://doi.org/10.1016/j.str.2006.10.004
Batie CJ, LaHaie E, Ballou DP (1987) Purification and characterization of phthalate oxygenase and phthalate oxygenase reductase from Pseudomonas cepacia. J Biol Chem 262:1510–1518
Boyd JM, Endrizzi JA, Hamilton TL, Christopherson MR, Mulder DW, Downs DM, Peters JW (2011) FAD binding by ApbE protein from Salmonella enterica: a new class of FAD-binding proteins. J Bacteriol 193:887–895. https://doi.org/10.1128/JB.00730-10
Ceja-Navarro JA, Vega FE, Karaoz U, Hao Z, Jenkins S, Lim HC, Kosina P, Infante F, Northen TR, Brodie EL (2015) Gut microbiota mediate caffeine detoxification in the primary insect pest of coffee. Nat Commun 6:7618. https://doi.org/10.1038/ncomms8618
Dash SS, Gummadi SN (2006) Catabolic pathways and biotechnological applications of microbial caffeine degradation. Biotechnol Lett 28:1993–2002. https://doi.org/10.1007/s10529-006-9196-2
Dash SS, Gummadi SN (2007) Optimization of physical parameters for biodegradation of caffeine by Pseudomonas sp.: a statistical approach. Am J Food Technol 2:21–29
Dash SS, Gummadi SN (2008) Inducible nature of the enzymes involved in catabolism of caffeine and related methylxanthines. J Basic Microbiol 48:227–233. https://doi.org/10.1002/jobm.200800004
Dawson WK, Jono R, Terada T, Shimizu K (2016) Electron transport in a dioxygenase-ferredoxin complex: long range charge coupling between the Rieske and non-heme iron center. PLoS One 11:e0162031. https://doi.org/10.1371/journal.pone.0162031
Gassner GT, Ludwig ML, Gatti DL, Correll CC, Ballou DP (1995) Structure and mechanism of the iron-sulfur flavoprotein phthalate dioxygenase reductase. FASEB J 9:1411–1418
Gokulakrishnan S, Chandraraj K, Gummadi SN (2007) A preliminary study of caffeine degradation by Pseudomonas sp. GSC 1182. Int J Food Microbiol 113:346–350. https://doi.org/10.1016/j.ijfoodmicro.2006.07.005
Green MR, Sambrook J, Sambrook J (2012) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Gummadi SN, Bhavya B (2011) Enhanced degradation of caffeine and caffeine demethylase production by Pseudomonas sp. in bioreactors under fed-batch mode. Appl Microbiol Biotechnol 91:1007–1017. https://doi.org/10.1007/s00253-011-3319-y
Gummadi S, Santhosh D (2006) How induced cells of Pseudomonas sp. increase the degradation of caffeine. Open Life Sci 1: https://doi.org/10.2478/s11535-006-0032-4
Haga T, Hirakawa H, Nagamune T (2013) Fine tuning of spatial arrangement of enzymes in a PCNA-mediated multienzyme complex using a rigid poly-L-proline linker. PLoS One 8:e75114. https://doi.org/10.1371/journal.pone.0075114
Karlsson A, Beharry ZM, Matthew Eby D, Coulter ED, Neidle EL, Kurtz DM, Eklund H, Ramaswamy S (2002) X-ray crystal structure of benzoate 1,2-dioxygenase reductase from Acinetobacter sp. strain ADP1. J Mol Biol 318:261–272. https://doi.org/10.1016/S0022-2836(02)00039-6
Kirksey TJ, Kwan S-W, Abell CW (1998) Arginine-42 and threonine-45 are required for FAD incorporation and catalytic activity in human monoamine oxidase B †. Biochemistry (Mosc) 37:12360–12366. https://doi.org/10.1021/bi9806910
Kweon O, Kim S-J, Baek S, Chae J-C, Adjei MD, Baek D-H, Kim Y-C, Cerniglia CE (2008) A new classification system for bacterial Rieske non-heme iron aromatic ring-hydroxylating oxygenases. BMC Biochem 9:11. https://doi.org/10.1186/1471-2091-9-11
Lin J, Zhou T, Ye K, Wang J (2007) Crystal structure of human mitoNEET reveals distinct groups of iron sulfur proteins. Proc Natl Acad Sci 104:14640–14645. https://doi.org/10.1073/pnas.0702426104
Marohnic CC, Barber MJ (2001) Arginine 91 is not essential for flavin incorporation in hepatic cytochrome b5 reductase. Arch Biochem Biophys 389:223–233. https://doi.org/10.1006/abbi.2001.2340
Nakamaru-Ogiso E, Yano T, Ohnishi T, Yagi T (2002) Characterization of the iron-sulfur cluster coordinated by a cysteine cluster motif (C XX C XXX C X 27 C) in the Nqo3 subunit in the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Thermus thermophilus HB-8. J Biol Chem 277:1680–1688. https://doi.org/10.1074/jbc.M108796200
Nathanson JA (1984) Caffeine and related methylxanthines: possible naturally occurring pesticides. Science 226:184–187
Phongsak T, Sucharitakul J, Thotsaporn K, Oonanant W, Yuvaniyama J, Svasti J, Ballou DP, Chaiyen P (2012) The C-terminal domain of 4-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii is an autoinhibitory domain. J Biol Chem 287:26213–26222. https://doi.org/10.1074/jbc.M112.354472
Retnadhas S (2014) Optimization of process conditions for biotransformation of caffeine to theobromine using induced whole cells of Pseudomonas sp. J Bioprocess Biotech. https://doi.org/10.4172/2155-9821.1000178
Sucharitakul J, Chaiyen P, Entsch B, Ballou DP (2005) The reductase of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii requires p-hydroxyphenylacetate for effective catalysis †. Biochemistry (Mosc) 44:10434–10442. https://doi.org/10.1021/bi050615e
Summers RM, Louie TM, Yu CL, Subramanian M (2011) Characterization of a broad-specificity non-haem iron N-demethylase from Pseudomonas putida CBB5 capable of utilizing several purine alkaloids as sole carbon and nitrogen source. Microbiology 157:583–592. https://doi.org/10.1099/mic.0.043612-0
Summers RM, Louie TM, Yu C-L, Gakhar L, Louie KC, Subramanian M (2012) Novel, highly specific N-demethylases enable bacteria to live on caffeine and related purine alkaloids. J Bacteriol 194:2041–2049. https://doi.org/10.1128/JB.06637-11
Summers RM, Seffernick JL, Quandt EM, Yu CL, Barrick JE, Subramanian MV (2013) Caffeine junkie: an unprecedented glutathione s-transferase-dependent oxygenase required for caffeine degradation by Pseudomonas putida CBB5. J Bacteriol 195:3933–3939. https://doi.org/10.1128/JB.00585-13
Summers RM, Mohanty SK, Gopishetty S, Subramanian M (2015) Genetic characterization of caffeine degradation by bacteria and its potential applications: genetics of bacterial caffeine degradation. Microb Biotechnol 8:369–378. https://doi.org/10.1111/1751-7915.12262
Wilks A, Black SM, Miller WL, Ortiz de Montellano PR (1995) Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase. Biochemistry (Mosc) 34:4421–4427. https://doi.org/10.1021/bi00013a034
Acknowledgements
The authors acknowledge IITM-SAIF and DST FIST facility for iron estimation by ICP-OES, DSC and CD spectroscopy. SR acknowledges UGC for fellowship.
Funding
This study was funded by the Department of Biotechnology, Ministry of Science and Technology, Government of India. Award Number: BT/PR11995/BBE/117/4/2014.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Rights and permissions
About this article
Cite this article
Retnadhas, S., Gummadi, S.N. Identification and characterization of oxidoreductase component (NdmD) of methylxanthine oxygenase system in Pseudomonas sp. NCIM 5235. Appl Microbiol Biotechnol 102, 7913–7926 (2018). https://doi.org/10.1007/s00253-018-9224-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-018-9224-x