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Structural Study of Soluble Ammonia Monooxygenase

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Structural Study of Soluble Ammonia Monooxygenase Structural study of soluble ammonia monooxygenase Trevor Obrinsky1, Alex McGurk1, Hasan DeMirci1,2 1PULSE Institute. 2Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA. +Contact: [email protected] Introduction 0.16 What makes Nitrosomonas europaea (N.e.) so 0.14 unique is its ability to generate all it’s required 0.12 energies from the two step conversion of 0.1 ammonia into nitrite. Ammonia monooxygenase (AMO) is the first of two enzymes in N.europaea 0.08 responsible for the oxidation of ammonia into nitrite. AMO catalyzes the conversion of ammonia 0.06 Absorbance Absorbance at 600 nm (Au) into the hydroxylamine intermediate. 0.04 AMO + - 0.02 NH3 + O2 + 2H + 2e NH2OH + H2O Fig. 3: Graph of absorbance, salt 0 0 20 40 60 80 100 120 concentration, and conductance during the Ammonia is useless to most organisms but this -0.02 elution of protein in a strong anion conversion into nitrite (part of the nitrogen cycle) Time (Hours) makes it accessible to other organisms that need exchange column. Absorbance peaks it for macromolecular synthesis. We are Fig. 1: Growth curve of represent potential subunits of AMO. interested in Nitrosomonas europaea and the Nitrosomonas europaea used to Bound protein was eluted across a 100 mL structure of ammonia monooxygenase due to its salt gradient from 0-1 M. crucial role in the nitrogen cycle. determine ideal time to harvest Keywords: Nitrosomonas europaea, ammonia N. europaea cells for maximum A B monooxygenase, nitrogen cycle AMO concentration Research Lee, et al. 150 mL N.e. stock culture Sirajuddin, et al. Fig. 4: A) Crystal structure of one protomer of membrane bound methane monooxygenase (pMMO) made up of 20 mL pmoB, pmoA, and pmoC. B) Crystal structure of soluble MMO (sMMO). MMO Fig. 2: Total cell paste from 12 and AMO are homologous enzymes. L of N. europaea growth 2 L N.e. at cultures. Cells are red because 1/100 dilution of iron heme groups. to determine Conclusions growth curve AMO Purification We determined the growth curve of N. europaea. Literature showed that harvesting cells in Lyse cells with 20 mL late exponential phase Emulsiflex maximized AMO in the cell. Future directions for this project include continuing the 2 L N.e. at purification process for the 1/100 dilution remaining N. europaea cell Ultracentrifuge (x6) for pellets, finding suitable production of crystallization conditions for pure AMO AMO, and using the crystals at a beam time to determine the crystal structure of AMO. FPLC with multiple columns to Acknowledgments isolate AMO Use of the Linac Coherent Light Source Purification of AMO (LCLS), SLAC National Accelerator Laboratory, is supported by the U.S. via FPLC Department of Energy, Office of Science, SDS-PAGE to confirm Office of Basic Energy Sciences under purification Contract No. DE-AC02-76SF00515. Date: 08/11/2016 .
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