Allen M. Orville

Allen Orville is a co-PI and a beamline scientist in the Macromolecular Crystallography Research Resource (PXRR) which provides facilities and support at the National Synchrotron Light Source for the benefit of outside and in-house investigators. The PXRR is supported by the NIH's National Center for Research Resources and the DOE Office of Biological and Environmental Research in its mission to create optimal facilities and environments for macromolecular structure determination by synchrotron X-ray diffraction. With a staff of about 24, the PXRR innovates new access modes such as FedEx crystallography, builds new facilities, currently on the X25 undulator, advances automation, develops remote participation software, collaborates with outside groups, teaches novice users, and supports visting investigators with 7day, 20 hours staff coverage.

In the News

• Sound, Light Sources and the Thrill of Glimpsing the Future  (February 28, 2013)
• Meet Allen Orville and Bo Yu  (February 21, 2012)
• Using Sound to Mount Microcrystals for X-ray Diffraction  (July 7, 2011)
• Getting More From Less: Correlated Single-Crystal Spectroscopy and X-ray Crystallography at the NSLS  (April 21, 2010)

Research Interests

Structural Basis of Enzyme Reaction Mechanisms, Protein Crystallography, Spectroscopy

Systems Biology of Microbe – Plant Symbiosis

• Symbiotic N₂-fixation by rhizobia within root nodules of leguminous plants
• High-throughput serial micro-crystallography with acoustic droplet ejection methods

Metalloenzymes and Flavoproteins

• Oxygen activation and reactivity in solution, and in low temperature crystals
• Methods for poising and studying enzyme reaction intermediates

Single-Crystal Spectroscopy Correlated with Synchrotron X-ray Crystallography

• X-ray diffraction and several types of spectroscopy from the same sample
• High-throughput structural biology applied to cofactor containing macromolecules

My broadest objectives include defining the structure-function relationships for a variety of metalloenzymes and flavoenzymes. Some of the enzymes, which are often studied in collaboration, transform either xenobiotic or naturally occurring, but recalcitrant chemicals. I like to emphasize x-ray crystallography and correlations of the structures with other complementary results. Some additional details of our progress can be found by following the appropriate links.

Briefly, my lab was the first to solve the crystal structure for any member of an effector protein in the multicomponent monooxygenase family ( T4moD). We were the first to demonstrate a C-terminal, domain-swapped dimer in the (alpha-beta)8-TIM barrel superfamily, which is the most common protein fold known ( XenA). My lab was the first to solve the crystal structure of a flavoprotein trapped during turnover of true substrates ( NAO-ES*). We were also the first to describe the structural basis for stoichiometric conversion of one O2 molecule into two H2O molecules by reaction of two NAD(P)H molecules in a flavoprotein ( NAD(P)H-Ox).

Most recently, we have solved the crystal structure of choline oxidase ( CHO) and have shown that it likely contains either an FAD C4a-OO(H) or C4a-O(H) intermediate, the first peroxy- or hydroxy-complex ever trapped in any flavoenzyme to date.

Synchrotron x-ray sources are vital to my research, and to the entire structural biology field. However, the intensity of synchrotron x-rays often produces radiation-induced alterations within the protein crystal. These may be interpreted as artifacts and largely overlooked, or they may provide novel structural insights. However, the latter is often difficult to establish due to the unfavorable bias inherent to the former. Spectroscopic data, especially if collected with a single-crystal microspectrophotometer from the crystal during diffraction studies, can be used to correlate spectroscopy, structure, and function. Moreover, these types of correlated studies are necessary to differentiate artifact from insight. This type of analysis is potentially applicable to a very large fraction of proteins, especially those that use redox-active cofactors, which often serve as an electron thermodynamic sink during a reaction cycle. However, electrons obtained during turnover of substrate molecule(s) do not differ from those obtained by photoreduction during x-ray diffraction data collection. In contrast, the proton inventory within the active site very likely does differ for substrate turnover versus photoreduction under cryogenic conditions. Consequently, low temperature photoreduction has been shown to trap reactive intermediates in crystals. Indeed, our studies of choline oxidase (CHO) appear to be a particularly good example of this phenomenon.

Selected Publications

• Daughtry K.D., Xiao Y., Stoner-Ma D., Cho E., Orville A.M., Liu P.*, and Allen K.N.
  Quaternary Ammonium Oxidative Demethylation: X-ray Crystallographic, Resonance Raman and UV-visible Spectroscopic Analysis of a Rieske-type Demethylase J. Am. Chem. Soc., 134(5):2823–2834 (2012).  PubMed
  PDB files: 3VCA, 3VCP 
• Orville A.M.
  Single-Crystal Spectroscopy Correlated with X-Ray Crystallography Provides Complementary Perspectives on Macromolecular Function
  Chapter 9 in Methods in Protein Biochemistry, edited by Harald Tschesche, ISBN 978-3-11-218934-4, DE GRUYTER, Berlin, Germany, pages 143-164 (2012).
• Berman L.E., Allaire M., Chance M.R., Hendrickson W.A., Héroux A., Jakoncic J., Liu Q., Orville A.M., Robinson H.H., Schneider D.K., Shi W., Soares A.S., Stojanoff V., Stoner-Ma D., and Sweet R.M.
  Optics Concept for a Pair of Undulator Beamlines for MX.
  Nuclear Instruments and Methods in Physics Research A, 649(1):131–135 (2011).  PubMed
• Orville A.M., Buono R., Cowan M., Héroux A., Schneider D.K., Shea-McCarthy G., Skinner J.M., Skinner M.J., Stoner-Ma D., and Sweet, R.M.
  Correlated Single-Crystal Electron Absorption Spectroscopy and X-ray Crystallography at NSLS Beamline X26-C.
  J. Synchrotron Radiation, 18:358–366 (2011).  PubMed
• Soares A.S., Engel M.A., Stearns R., Datwani S., Olechno J., Ellson R., Skinner J.M., Allair M., and Orville A.M.
  Acoustically Mounted Microcrystals Yield High Resolution X-ray Structures.
  a Rapid Report in Biochemistry, 50:4399–4401 (2011).  PubMed
  PDB files: 3RTO,  
• Stoner-Ma D., Skinner J.M., Héroux A., Schneider D.K., Sweet R.M. Orville A.M.
  Single-Crystal Raman Spectroscopy and X-ray Crystallography at Beamline X26-C of the NSLS.
  J. Synchrotron Rad., 18:37–40 (2011).  PubMed
• Finnegan S., Yuan H., Wang Y-F., Orville A.M., Weber I.T., and Gadda G.
  Structural and Kinetic Studies on the Ser101Ala Variant of Choline Oxidase.
  Archives of Biochemistry and Biophysics, 501:207–213, DOI:10.1016/j.abb.2010.06.014,  (2010).  PubMed
  PDB files: 3NNE,  
• Yi J., Orville A.M., Skinner J.M., Skinner M.J., and Richter-Addo, G.B.
  Synchrotron X-ray-Induced Photoreduction of Ferric Myoglobin Nitrite Crystals Gives the Ferrous Derivative with Retention of the O-bonded Nitrite Ligand
  Rapid Report in Biochemistry, 49:5969–5971, PMID: 20568729, DOI: 10.1021/bi100801g,(2010).   PubMed
  PDB files: 3LR7,  
• Héroux A., Bozinovski D.M., Valley M.P., Fitzpatrick P.F., and Orville A.M.
  Crystal Structures of Intermediates in the Nitroalkane Oxidase Reaction
  Biochemistry, 48(15):3407-3416 (2009).  PubMed
  PDB files: 3D9D, 3D9E, 3D9F, 3D9G ,  
• Major D.T., Héroux A., Orville A.M., Valley M.P., Fitzpatrick P.F., and Gao J.
  Differential quantum tunneling contributions in nitroalkane oxidase catalyzed and the uncatalyzed proton transfer reaction. Proc. Nat. Acad. Sci. USA, 106:20734–20739 (2009), PDB ID code 3FCJ  PubMed
• Orville A.M., Lountos G.T., Finnegan S., Gadda G., and Prabhakar R.
  Crystallographic, Spectroscopic, and Computational Analysis of a Flavin-C4a-Oxygen Adduct in Choline Oxidase
  Biochemistry, 48(4):720–728 (2009).  PubMed
  PDB file: 2JBV,
• Gadda G., Pennatil A., Francis K., Quaye O., Yuan H., Rungsrisuriyachai K., Finnegan S., Mijatovic S., Nguyen T., and Orville A.M.
  Hydride transfer made easy in the oxidation of alcohols catalyzed by choline oxidase.
  Flavins and Flavoproteins 2008: Proceedings of the Sixteenth International Symposium, Palacio de Congresos, Jaca, Spain, June 8-13, 2008, S. Frago, C. Gomez-Moreno, and M. Medina, Editors, Prensas Universitarias de Zaragoza, Spain, pp. 309-314 (2008).

• Quaye O., Lountos G.T., Fan F., Orville A.M. and Gadda G.
  Role of Glu312 in binding and positioning of the substrate for hydride transfer reaction in choline oxidase.
  Biochemistry, 47(1):243-256 (2008).  PubMed
  PDB file: 2JBV,   Cover: The crystal structure of choline oxidase provides mechanistic insights and reveals a novel FAD C4a-adduct. The solvent accessible surfaces of the homodimeric enzyme are colored to illustrate the dimer interface and the major domains within one subunit. The FAD binding (blue and the substrate binding (green) domains are partially separated by a loop (orange) that sequesters the active site within the interior of the enzyme.
• Pau MYM, Davis MI, Orville AM, Lipscomb JD, and Solomon EI.
  Spectroscopic and Electronic Structure Study of the Enzyme-Substrate Complex of Intradiol Dioxygenases: Substrate Activation by a High-Spin Ferric Non-heme Iron Site.
  J Am Chem Soc., 129:1944-1958 (2007).  PubMed

• Lountos G.T., Jiang R., Wellborn W.B., Thaler T.L., Bommarius A.S. and Orville A.M.
  The Crystal Structure of NAD(P)H Oxidase from Lactobacillus sanfranciscensis: Insights into the Conversion of O2 into Two Water Molecules by the Flavoenzyme.
  Biochemistry, 45:9648-9659 (2006).   PubMed
  PDB file: 2CDU

• Nagpal A., Valley M.P., Fitzpatrick P.F. and Orville A.M.
  Crystal Structures of Nitroalkane Oxidase: Insights into the Reaction Mechanism from a Covalent Complex of the Flavoenzyme Trapped during Turnover.
  Biochemistry, 45:1138-1150 (2006).   PubMed
  PDB files: 2C12   2C0U  
• Orville A.M., Nagpal A., Manning L., Blehert D.S., Valley M.P., Chambliss G.H., Fox B.G. and Fitzpatrick P.F.
  Structural Perspective on Nitrite Elimination of Organic Nitrochemicals by Flavoenzymes.
  in Flavins and Flavoproteins 2005, (T. Nishino, R. Miura, M. Tanokura, K. Fukui, edts) ARchiTect Inc., 827-840 (2005).
• Fitzpatrick P.F., Valley M.P., Gadda G., Nagpal A. and Orville A.M.
  The Mechanism of Nitroalkane Oxidase.
  in Flavins and Flavoproteins 2005, (T. Nishino, R. Miura, M. Tanokura, K. Fukui, edts) ARchiTect Inc., 59-69 (2005).
• Nagpal A., Valley M.P., Fitzpatrick P.F. and Orville A.M.
  Crystal structures of nitroalkane oxidase: High resolution data collection strategy for long cell edged crystals.
  NSLS Science Highlights, 2004 NSLS Activity Report (2005).
• Fitzpatrick P.F., Orville A.M., Nagpal A. and Valley M.P.
  Nitroalkane oxidase, a carbanion-forming flavoprotein homologous to acyl-CoA dehydrogenase.
  Arch Biochem Biophys., 433:157-165 (2005).   PubMed
  

• Lountos G.T., Mitchell K.H., Studts J.M., Fox B.G. and Orville A.M.
  Crystal structures and functional studies of T4moD, the toluene 4-monooxygenase catalytic effector protein.
  Biochemistry, 44:7131-7142 (2005).   PubMed
  PDB files: 2BF2   2BF3   2BF5  
• Lountos G.T., Riebel B.R., Wellborn W.B., Bommarius A.S., and Orville A.M.
  Crystallization and preliminary analysis of a water-forming NADH oxidase from Lactobacillus sanfranciscensis.
  Acta Crystallography, D60:2044�2047 (2004).   PubMed
• Nagpal A., Valley M.P., Fitzpatrick P.F. and Orville A.M.
  Crystallization and preliminary analysis of active nitroalkane oxidase in three crystal forms.
  Acta Crystallography, D60:1456-1460 (2004).   PubMed
• Orville A.M., Manning L., Blehert D.S., Fox B.G. and Chambliss G.H.
  Crystallization and preliminary analysis of xenobiotic reductase B and ligand complexes from Pseudomonas fluorescens I-C.
  Acta Crystallography, D60:1289-1291 (2004).   PubMed
• Orville A.M., Manning L., Blehert D.S., Studts J.M., Fox B.G. and Chambliss G.H.
  Crystallization and preliminary analysis of xenobiotic reductase A and ligand complexes from Pseudomonas putida II-B.
  Acta Crystallography, D60:957-961 (2004).   PubMed
• Orville A.M., Studts J.M., Lountos G.T., Mitchell K.H. and Fox B.G.
  Crystallization and preliminary analysis of natural and N-terminal truncated isoforms of toluene-4-monooxygenase catalytic effector protein.
  Acta Crystallography, D59:572-575 (2003).   PubMed
• Wasinger E.C., Davis M.I., Pau M., Orville A.M., Zaleski J.M., Hedman B., Lipscomb J.D., Hodgson K.O. and Solomon E.I.
  Spectroscopic studies of the effect of ligand donor strength on the Fe-NO bond in intradiol dioxygenases.
  Inorganic Chem., 42:365-376 (2003).   PubMed
• Davis M.I., Orville A.M., Neese F., Zaleski J.M., Lipscomb J.D. and Solomon E.I.
  Spectroscopic and theoretical studies of protocatechuate 3,4-dioxygenase: Nature of the tyrosinate-Fe(III) bonds and their contribution to reactivity
  J Am Chem Soc., 124:602-614 (2002).

• Lowther W.T., Orville A.M., Madden D.T., Lim S., Rich D.H. and Matthews B.W.
  E. coli methionine aminopeptidase: Implications of crystallographic analyses of the native, mutant and inhibited enzymes for the mechanism of catalysis.
  Biochemistry, 38:7678-7688 (1999).   PubMed
  PDB files: 4MAT   2MAT   3MAT  
• Davis M.I., Wasinger E.C., Westre T.E., Zaleski J.M., Orville A.M., Lipscomb J.D., Hedman B., Hodgson K.O. and Solomon E.I.
  Spectroscopic investigation of reduced protocatechuate 3,4-dioxygenase: charge induced alterations in the active site iron coordination environment.
  Inorganic Chem., 38:3676-3683 (1999).
• Lowther W.T., McMillen D.A., Orville A.M. and Matthews B.W.
  The anti-angiogenic agent fumagillin covalently modifies a conserved active-site histidine in the Escherichia coli methionine aminopeptidase.
  Proc Natl Acad Sci USA, 95:12153-12157 (1998).   PubMed   Full Text

• Frazee R.W., Orville A.M., Dolbeare K.B., Hong Y., Ohlendorf D.H. and Lipscomb J.D.
  The axial tyrosinate-Fe3+ ligand in protocatechuate 3,4-dioxygenase influences substrate binding and product release: evidence for new reaction cycle intermediates.
  Biochemistry, 37:2131-2144 (1998).   PubMed
  PDB file: 3PCD  
• Lipscomb J.D., Orville A.M., Frazee R.W., Miller M.A. and Ohlendorf D.H.
  Fundamentally divergent strategies for catecholic dioxygenases.
  in; Oxygen Homeostasis and Its Dynamics, (eds: Y. Ishimura, H. Shimada, and M. Suematsu) Springer-Verlag Tokyo, 263-275 (1998).
• Lipscomb J.D., Orville A.M., Frazee R.W., Dolbeare K.B., Elango N. and Ohlendorf D.H.
  Intermediates in non-heme iron intradiol dioxygenase catalysis.
  in; Spectroscopic Methods in Bioinorganic Chemistry, ACS Symposium Series 692, (eds: E.I. Solomon and K.O. Hodgson) ACS Publishing, Washington, DC, 387-402 (1998).
• Orville A.M., Lipscomb J.D. and Ohlendorf D.H.
  Probing the reaction mechanism of protocatechuate 3,4-dioxygenase with X-ray crystallography.
  in: Oxygen Homeostasis and Its Dynamics, (eds: Y. Ishimura, H. Shimada, and M. Suematsu) Springer-Verlag Tokyo, 282-288 (1998).
• Orville A.M. and Lipscomb J.D.
  Cyanide and nitric oxide binding to reduced protocatechuate 3,4-dioxygenase: Insight into the basis for order dependent ligand binding by intradiol catecholic dioxygenases.
  Biochemistry, 36:14044-14055 (1997).   PubMed

• Elgren T.E., Orville A.M., Kelly K.A., Lipscomb J.D., Ohlendorf D.H. and Que L. Jr.
  Crystal structure and resonance raman studies of protocatechuate 3,4-dioxygenase complexed with 3,4-dihydroxyphenylacetate.
  Biochemistry, 36:11504-11513 (1997).   PubMed
  PDB file: 3PCN  

• Orville A.M., Lipscomb J.D. and Ohlendorf D.H.
  Crystal structures of substrate and substrate analog complexes of protocatechuate 3,4-dioxygenase: Endogenous ligand displacement in response to substrate binding.
  Biochemistry, 36:10052-10066 (1997).   PubMed
  PDB files: 3PCJ   3PCK   3PCL   3PCM   3PCA  

• Orville A.M., Elango N., Lipscomb J.D. and Ohlendorf D.H.
  Structures of competitive inhibitor complexes of protocatechuate 3,4-dioxygenase: multiple exogenous ligand orientations within the active site.
  Biochemistry, 36:10039-10051 (1997).   PubMed
  PDB files: 3PCI   3PCH   3PCF   3PCG   3PCE   3PCC   3PCB  
• Shu L., Chiou Y.-M., Orville A.M., Miller M.A., Lipscomb J.D. and Que L. Jr.
  X-ray absorption spectroscopic studies of the Fe(II) active site of catechol 2,3-dioxygenase: implications for the extradiol cleavage mechanism.
  Biochemistry, 33:6649-6659 (1995).   PubMed
  

• Ohlendorf D.H., Orville A.M. and Lipscomb J.D.
  Structure of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa at 2.15 Å resolution.
  J Mol Biol, 244:586-608 (1994).   PubMed
  PDB file: 2PCD   Jmol viewer
  
• Earhart C.A., Radhakrishnan R., Orville A.M., Lipscomb J.D. and Ohlendorf D.H.
  Preliminary crystallographic study of protocatechuate 3,4-dioxygenase from Brevibacterium fuscum.
  J Mol Biol., 236:374-376 (1994).   PubMed
• Orville A.M. and Lipscomb J.D.
  Simultaneous binding of nitric oxide and isotopically labeled substrates or inhibitors by reduced protocatechuate 3,4-dioxygenase.
  J Biol Chem., 268:8596-8607 (1993).   PubMed   Full Text
• Paulsen K.E., Stankovich M.T. and Orville A.M.
  Electron paramagnetic resonance spectroelectrochemcal titration.
  in: Methods in Enzymology, Vol. 227, (eds. J.F. Riordan and B.L. Vallee), Academic Press Inc., New York, 396-411 (1993).   PubMed
• Paulsen K.E., Orville A.M., Frerman F.E., Lipscomb J.D. and Stankovich M.T.
  The redox properties of electron-transfer flavoprotein ubiquinone oxidoreductase as determined by EPR-spectroelectrochemistry.
  Biochemistry, 31:11755-11761 (1992).   PubMed
• Siu D.C.-T., Orville A.M., Lipscomb J.D., Ohlendorf D.H. and Que L. Jr.
  Resonance Raman studies of the protocatechuate 3,4-dioxygenase from Brevibacterium fuscum.
  Biochemistry, 31:10443-10448 (1992).   PubMed
• Orville A.M., Chen V.C., Kriauciunas A., Harpel M.R., Fox B.G., Münck E. and Lipscomb J.D.
  Thiol ligation of the active site Fe2+ of isopenicillin N synthase derives from substrate rather than endogenous cysteine: Spectroscopic studies of site-specific Cys-Ser mutated enzymes.
  Biochemistry, 31, 4602-4612 (1992).   PubMed
• Lipscomb J.D. and Orville A.M.
  Mechanistic aspects of dihydroxybenzoate dioxygenases.
  in: Metal Ions in Biological Systems, Vol. 28, (eds. H. Sigel and A. Sigel), Marcel Dekker Inc., New York, 243-298 (1992).
• Paulsen K.E., Orville A.M., Frerman F.E., Stankovich M.T. and Lipscomb J.D.
  EPR-spectroelectrochemistry of mammalian electron-transfer flavoprotein-ubiquinone oxidoreductase.
  in: Progress in Clinical and Biological Research, Vol. 357, (eds. P.M. Coates, and K. Tanaka), Wiley-Liss Inc., New York, 69-73 (1992).
• Mabrouk P.M., Orville A.M., Lipscomb J.D. and Solomon E.I.
  Variable-temperature variable-field magnetic circular dichroism studies of the Fe(II) active site in metapyrocatechase: Implications for the molecular mechanism of extradiol dioxygenases.
  J Am Chem Soc., 113:4053-4061 (1991).  
• True A.E., Orville A.M., Pearce L.L., Lipscomb J.D. and Que L. Jr.
  An EXAFS study of the interaction of substrate with the ferric active site of protocatechuate 3,4-dioxygenase.
  Biochemistry, 29:10847-10854 (1990).   PubMed
• Que L. Jr., True A.E., Pearce L.L., Orville A.M. and Lipscomb J.D.
  EXAFS studies of protocatechuate 3,4-dioxygenase from Brevibacterium fuscum.
  Yamada Conference XXVII, International Symposium on Oxygenases and Oxygen Activation, Kyoto, Japan, 99-102 (1990).
• Lipscomb J.D., Orville A.M., Ohlendorf D.H. and Weber P.C.
  Structure and mechanism of protocatechuate 3,4-dioxygenase.
  Yamada Conference XXVII, International Symposium on Oxygenases and Oxygen Activation, Kyoto, Japan, 95-98 (1990).
• Orville A.M., Harpel M.R. and Lipscomb J.D.
  Synthesis of [17O or 18O]-enriched dihydroxy aromatic compounds.
  in: Methods in Enzymology, Vol. 188, (ed. M. Lidstrom), Academic Press, New York, 107-115 (1990).   PubMed
• Arciero D.M., Orville A.M. and Lipscomb J.D.
  Protocatechuate 4,5-dioxygenase from Pseudomonas testosteroni.
  in: Methods in Enzymology, Vol. 188, (ed. M. Lidstrom), Academic Press, New York, 89-95 (1990).   PubMed
• Whittaker J.W., Orville A.M. and Lipscomb J.D.
  Protocatechuate 3,4-dioxygenase from Brevibacterium fuscum.
  in: Methods in Enzymology, Vol. 188, (ed. M. Lidstrom), Academic Press, New York, 82-88 (1990).   PubMed
• Chen V.J., Orville A.M., Harpel M.R., Frolik C.A., Surerus K.K., Münck E., and Lipscomb J.D.
  Spectroscopic studies of isopenicillin N synthase: A mononuclear nonheme Fe2+ oxidase with metal coordination sites for small molecules and substrate.
  J Biol Chem., 264:21677-21681 (1989).   PubMed   Full Text
• Orville A.M. and Lipscomb J.D.
  Binding of isotopically labeled substrates, inhibitors and cyanide by protocatechuate 3,4-dioxygenase.
  J Biol Chem., 264:8791-8801 (1989).   PubMed   Full Text
• Lipscomb J.D., Whittaker J.W., Arciero D.M., Orville A.M. and Wolgel S.A.
  Mechanisms of catecholic dioxygenases.
  in: Microbial Metabolism and the Carbon Cycle, (eds. S.R. Hagedorn, R.S. Hanson, and D.A., Kunz), Harwood Academic Publishers, NY, 259-282 (1988).
• Arciero D.M., Orville A.M and Lipscomb J.D.
  17-O-Water and nitric oxide binding by protocatechuate 4,5-dioxygenase and catechol 2,3-dioxygenase: Evidence for binding of exogenous ligands to the active site Fe(II) of extradiol dioxygenase.
  J Biol Chem., 260:14035-14044 (1985).   PubMed   Full Text