Allen M. Orville Biology Department, 463 Brookhaven National Laboratory Upton, NY 11973-5000 tel: (631) 344-4739 lab: (631) 344-5726 fax: (631) 344-2741 amorv@bnl.gov

Allen Orville is a beam line 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.

Research Interests:	Structural basis of enzyme reaction mechanisms, protein crystallography, spectroscopy
Metalloenzymes:	a) Catabolic pathways of aromatic hydrocarbons b) Biosynthetic pathways of antibiotics containing halogens and/or R-NO2
Flavoenzymes:	a) Reductive transformations of nitroesters and nitroaromatics b) Oxidative transformations of nitroalkanes c) Oxygen activation by flavoenzyme oxidases
Synchrotron X-ray Crystallography:	a) Diffraction and spectroscopy from the same crystal b) Trapping enzyme reaction intermediates

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.

Publications:

Note: PDB files referenced below can be viewed with

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 GT, Jiang R, Wellborn WB, Thaler TL, Bommarius AS, and Orville AM. 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   Jmol viewer
	Nagpal A, Valley MP, Fitzpatrick PF, and Orville AM. 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   Jmol viewer

Orville AM, Nagpal A, Manning L, Blehert DS, Valley MP, Chambliss GH, Fox BG, and Fitzpatrick PF. 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 PF, Valley MP, Gadda G, Nagpal A, and Orville AM. 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 MP, Fitzpatrick PF, and Orville AM. Crystal structures of nitroalkane oxidase: High resolution data collection strategy for long cell edged crystals. NSLS Science Highlights, 2004 NSLS Activity Report (2005).

Fitzpatrick PF, Orville AM, Nagpal A, and Valley MP. Nitroalkane oxidase, a carbanion-forming flavoprotein homologous to acyl-CoA dehydrogenase. Arch Biochem Biophys 433 157-165 (2005).   PubMed

Lountos GT, Mitchell KH, Studts JM, Fox BG, and Orville AM. 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   Jmol viewer

Lountos GT, Riebel BR, Wellborn WB, Bommarius AS, and Orville AM. Crystallization and preliminary analysis of a water-forming NADH oxidase from Lactobacillus sanfranciscensis. Acta Crystallography. D60, 2044–2047 (2004).   PubMed

Nagpal A, Valley MP, Fitzpatrick PF, and Orville AM. Crystallization and preliminary analysis of active nitroalkane oxidase in three crystal forms. Acta Crystallography D60 1456-1460 (2004).   PubMed

Orville AM, Manning L, Blehert DS, Fox BG, and Chambliss GH. Crystallization and preliminary analysis of xenobiotic reductase B and ligand complexes from Pseudomonas fluorescens I-C. Acta Crystallography D60, 1289-1291 (2004).   PubMed

Orville AM, Manning L, Blehert DS, Studts JM, Fox BG, and Chambliss GH. Crystallization and preliminary analysis of xenobiotic reductase A and ligand complexes from Pseudomonas putida II-B. Acta Crystallography D60, 957-961 (2004).   PubMed

Orville AM, Studts JM, Lountos GT, Mitchell KH, and Fox BG. 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 EC, Davis MI, Pau M, Orville AM, Zaleski JM, Hedman B, Lipscomb JD, Hodgson KO, Solomon EI. 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 MI, Orville AM, Neese F, Zaleski JM, Lipscomb JD, and Solomon EI. 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 WT, Orville AM, Madden DT, Lim S, Rich DH, and Matthews BW. 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   Jmol viewer

Davis MI, Wasinger EC, Westre TE, Zaleski JM, Orville AM, Lipscomb JD, Hedman B, Hodgson KO, and Solomon EI. 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 WT, McMillen DA, Orville AM, and Matthews BW. 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 RW, Orville AM, Dolbeare KB, Hong Y, Ohlendorf DH, and Lipscomb JD. 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   Jmol viewer

Lipscomb JD, Orville AM, Frazee RW, Miller MA, and Ohlendorf DH. 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 JD, Orville AM, Frazee RW, Dolbeare KB, Elango N, and Ohlendorf DH. 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 AM, Lipscomb JD, and Ohlendorf DH. 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 AM and Lipscomb JD. 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 TE, Orville AM, Kelly KA, Lipscomb JD, Ohlendorf DH, 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   Jmol viewer
	Orville AM, Lipscomb JD, and Ohlendorf DH. 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   Jmol viewer
	Orville AM, Elango N, Lipscomb JD, and Ohlendorf DH. 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   Jmol viewer

Shu L, Chiou Y-M, Orville AM, Miller MA, Lipscomb JD, 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 DH, Orville AM, and Lipscomb JD. 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 CA, Radhakrishnan R, Orville AM, Lipscomb JD, and Ohlendorf DH. Preliminary crystallographic study of protocatechuate 3,4-dioxygenase from Brevibacterium fuscum. J Mol Biol 236, 374-376 (1994).   PubMed

Orville AM and Lipscomb JD. 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 KE, Stankovich MT, and Orville AM. 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 KE, Orville AM, Frerman FE, Lipscomb JD, and Stankovich MT. The redox properties of electron-transfer flavoprotein ubiquinone oxidoreductase as determined by EPR-spectroelectrochemistry. Biochemistry, 31, 11755-11761 (1992).   PubMed

Siu DC-T, Orville AM, Lipscomb JD, Ohlendorf DH, and Que L Jr. Resonance Raman studies of the protocatechuate 3,4-dioxygenase from Brevibacterium fuscum. Biochemistry, 31, 10443-10448 (1992).   PubMed

Orville AM, Chen VC, Kriauciunas A, Harpel MR, Fox BG, Münck E, and Lipscomb JD. 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 JD and Orville AM. 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 KE, Orville AM, Frerman FE, Stankovich MT, and Lipscomb JD. 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 PM, Orville AM, Lipscomb JD, and Solomon EI. 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 AE, Orville AM, Pearce LL, Lipscomb JD, 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 AE, Pearce LL, Orville AM, and Lipscomb JD. 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 JD, Orville AM, Ohlendorf DH, and Weber PC. Structure and mechanism of protocatechuate 3,4-dioxygenase. Yamada Conference XXVII, International Symposium on Oxygenases and Oxygen Activation, Kyoto, Japan, 95-98 (1990).

Orville AM, Harpel MR, and Lipscomb JD. 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 DM, Orville AM, and Lipscomb JD. 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 JW, Orville AM, and Lipscomb JD. 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 VJ, Orville AM, Harpel MR, Frolik CA, Surerus KK, Münck E, and Lipscomb JD. 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 AM and Lipscomb JD. Binding of isotopically labeled substrates, inhibitors and cyanide by protocatechuate 3,4-dioxygenase. J Biol Chem, 264, 8791-8801 (1989).   PubMed   Full Text

Lipscomb JD, Whittaker JW, Arciero DM, Orville AM, and Wolgel SA. 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 DM, Orville AM, and Lipscomb JD. 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

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