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Studying the Role of Histidine-133 in Class I Nitronate Monooxygenase from Pseudomonas Aeruginosa PAO1 by SiteDirected Mutagenesis

Undergraduate #140
Discipline: Chemistry and Chemical Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)

Rachel Kyeremaa - University of Georgia
Co-Author(s): Dan Su and Giovanni Gadda, Georgia State University, GA



Propionate 3-nitronate (P3N) is a highly toxic nitro-compound found in plants and fungi. The toxic compound exist in equilibrium with 3-Nitropropionic acid (3-PNPA) at a physiological PH. The Nitro alkanes contains nitro group that display a pka of the alpha carbon much lower than actual alkane’s pka valve of 30-40. The toxic compound (P3N) leads to various neurological disorders due to its capability to irreversibly inhibit mitochondrial succinate dehydrogenase in the Krebs cycle. Cases of human and stock poisoning by P3N have been documented as there is currently no antidote that has been discovered. Nitronate monooxygenase (NMO) from Pseudomonas aeruginosa PAO1 (PaNMO), the first NMO identified in bacteria, was shown to oxidize P3N with the cofactor of FMN. The presence of neutral and anionic flavin semiquinones was detected during enzyme turnover with P3N as substrate using a stopped flow spectrophotometer equipped with a photodiode array, indicating one electron transfer involved in catalysis. Two forms of flavin semiquinones display a pKa of 6.3 ± 0.1 in anaerobic reduction of PaNMO with P3N. In the crystal structure of PaNMO, histidine-133 is located 4.1 Å from N1 atom of FMN, and it is proposed to play a role in modulating the pKa of flavin semiquinones. To test this proposal, histidine-133 was mutated to phenylalanine, tyrosine, and asparagine. The formation of the flavin species during catalysis can then be monitored to probe the significance of histidine-133 for flavin semiquinones stabilization. The molecular biology aspect of site-directed mutagenesis of histidine-133 to phenylalanine, tyrosine, and asparagine was successfully done. We were able to purify these proteins but unable to observe the role of histidine-133 in the active site of the enzyme and how it also affect the role of modulating redox potentials of different FMN redox states due to limited time. Future studies will focus on Kinetics and spectroscopic characterization of mutants in comparison with wild type to study the importance of histidine in the active site.

Reference: Francis K, Nishino SF, Spain JC, Gadda G. Arch. Biochem. Biophys. 2012 May; 521(1-2):84-9.
Salvi F, Agniswamy J, Yuan H, Vercammen K, Pelicaen R, Cornelis P, Spain J, Gadda G. J. Biol. Chem. 2014 Jul 7.
Unpublished data from Smitherman C and Gadda G. Stankovich MT, Schopfer LM and Massey V. J. Biol. Chem. 1978, 253:49714979.

Funder Acknowledgement(s): NSF MCB-1121695 (G.G.) / I thank Dr. Giovanni Gadda (P.I) for giving me the privilege to participate in this project and also allowing me to work in his lab. I also thank Dan Su my student mentor for his patient and support during my research and all the Ph.D students in the lab especially Crystal Lynn Smitherman. Funding was provided by NSF MCB-1121695 (G.G.).

Faculty Advisor: Giovanni Gadda,

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This material is based upon work supported by the National Science Foundation (NSF) under Grant No. DUE-1930047. Any opinions, findings, interpretations, conclusions or recommendations expressed in this material are those of its authors and do not represent the views of the AAAS Board of Directors, the Council of AAAS, AAAS’ membership or the National Science Foundation.

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