title: NMR crystallography of enzyme active sites: probing chemically detailed, three-dimensional structure in tryptophan synthase.
abstract: NMR crystallography--the synergistic combination of X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry--offers unprecedented insight into three-dimensional, chemically detailed structure. Initially, researchers used NMR crystallography to refine diffraction data from organic and inorganic solids. Now we are applying this technique to explore active sites in biomolecules, where it reveals chemically rich detail concerning the interactions between enzyme site residues and the reacting substrate. Researchers cannot achieve this level of detail from X-ray, NMR,or computational methodologies in isolation. For example, typical X-ray crystal structures (1.5-2.5 Å resolution) of enzyme-bound intermediates identify possible hydrogen-bonding interactions between site residues and substrate but do not directly identify the protonation states. Solid-state NMR can provide chemical shifts for selected atoms of enzyme-substrate complexes, but without a larger structural framework in which to interpret them only empirical correlations with local chemical structure are possible. Ab initio calculations and molecular mechanics can build models for enzymatic processes, but they rely on researcher-specified chemical details. Together, however, X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry can provide consistent and testable models for structure and function of enzyme active sites: X-ray crystallography provides a coarse framework upon which scientists can develop models of the active site using computational chemistry; they can then distinguish these models by comparing calculated NMR chemical shifts with the results of solid-state NMR spectroscopy experiments. Conceptually, each technique is a puzzle piece offering a generous view of the big picture. Only when correctly pieced together, however, can they reveal the big picture at the highest possible resolution. In this Account, we detail our first steps in the development of NMR crystallography applied to enzyme catalysis. We begin with a brief introduction to NMR crystallography and then define the process that we have employed to probe the active site in the β-subunit of tryptophan synthase with unprecedented atomic-level resolution. This approach has resulted in a novel structural hypothesis for the protonation state of the quinonoid intermediate in tryptophan synthase and its surprising role in directing the next step in the catalysis of L-Trp formation.
authors.affiliation: Department of Chemistry and ‡Department of Biochemistry, University of California , Riverside, California 92521, United States.
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Mueller
authors.forename: Michael F
authors.initials: MF
authors.lastname: Dunn
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011789:Quantum Theory; D014367:Tryptophan Synthase / Q000737:chemistry*
chemical_list: D014367:Tryptophan Synthase
references.citation: J Am Chem Soc. 2003 Oct 1;125(39):11784-5
references.pmid: 14505377
references.citation: J Am Chem Soc. 2011 Jan 12;133(1):4-7
references.pmid: 21142052
references.citation: Arch Biochem Biophys. 2012 Mar 15;519(2):154-66
references.pmid: 22310642
references.citation: Science. 1993 Jun 4;260(5113):1491-6
references.pmid: 8502992
references.citation: J Phys Chem A. 2010 Sep 30;114(38):10435-42
references.pmid: 20815383
references.citation: J Phys Chem B. 2007 Apr 19;111(15):3869-76
references.pmid: 17388551
references.citation: J Biol Chem. 1989 Sep 25;264(27):15774-80
references.pmid: 2506170
references.citation: Nature. 1993 Sep 23;365(6444):325-7
references.pmid: 8377823
references.citation: J Am Chem Soc. 2012 Mar 21;134(11):5006-9
references.pmid: 22393876
references.citation: Biochemistry. 2002 Aug 6;41(31):9982-90
references.pmid: 12146962
references.citation: J Am Chem Soc. 2010 Mar 3;132(8):2564-6
references.pmid: 20136091
references.citation: Biochemistry. 1990 Sep 18;29(37):8598-607
references.pmid: 2271543
references.citation: Curr Opin Struct Biol. 2007 Oct;17(5):617-22
references.pmid: 17964133
references.citation: Chembiochem. 2008 May 5;9(7):1024-8
references.pmid: 18351684
references.citation: J Biomol NMR. 2011 May;50(1):19-33
references.pmid: 21305337
references.citation: J Am Chem Soc. 2005 Jul 27;127(29):10365-70
references.pmid: 16028949
references.citation: Acta Crystallogr D Biol Crystallogr. 1998 Nov 1;54(Pt 6 Pt 1):1119-31
references.pmid: 10089488
references.citation: Proc Natl Acad Sci U S A. 2007 Feb 13;104(7):2080-5
references.pmid: 17287353
references.citation: Proc Natl Acad Sci U S A. 2011 Oct 11;108(41):16974-9
references.pmid: 21969532
references.citation: Annu Rev Phys Chem. 2002;53:349-78
references.pmid: 11972012
references.citation: J Am Chem Soc. 2007 Apr 11;129(14):4440-55
references.pmid: 17371021
references.citation: Biochim Biophys Acta. 2011 Nov;1814(11):1447-58
references.pmid: 21354475
references.citation: J Biol Chem. 1992 Jun 25;267(18):13028-38
references.pmid: 1618800
references.citation: J Biol Chem. 1988 Nov 25;263(33):17857-71
references.pmid: 3053720
references.citation: Phys Chem Chem Phys. 2006 Jan 7;8(1):137-43
references.pmid: 16482253
references.citation: J Am Chem Soc. 2002 Dec 4;124(48):14450-9
references.pmid: 12452721
references.citation: Proc Natl Acad Sci U S A. 1966 Apr;55(4):712-6
references.pmid: 5219675
references.citation: J Am Chem Soc. 2006 Feb 8;128(5):1547-52
references.pmid: 16448125
references.citation: Biochemistry. 1992 Apr 21;31(15):3831-9
references.pmid: 1567839
references.citation: Trends Biochem Sci. 1997 Jan;22(1):22-7
references.pmid: 9020588
references.citation: Trends Biochem Sci. 2008 Jun;33(6):254-64
references.pmid: 18486479
references.citation: Adv Enzymol Relat Areas Mol Biol. 1979;49:127-86
references.pmid: 400853
references.citation: Biochemistry. 1988 Jul 12;27(14):4966-70
references.pmid: 3167024
references.citation: Biochemistry. 2007 Jul 3;46(26):7740-53
references.pmid: 17559232
references.citation: Chem Rev. 2005 Aug;105(8):2999-3093
references.pmid: 16092826