Search Menu
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information
Search Scholarly Publications

Title: Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate

Journal Article · · Proceedings of the National Academy of Sciences of the United States of America
 [1];  [1];  [2];  [1];  [1]; ORCiD logo [3]; ORCiD logo [4]; ORCiD logo [1];  [1];  [1];  [5]; ORCiD logo [5]; ORCiD logo [5]; ORCiD logo [1]; ORCiD logo [6];  [7];  [7];  [7]; ORCiD logo [5]; ORCiD logo [1] more »;  [8];  [1]; ORCiD logo [3];  [1]; ORCiD logo [1] « less
  1. Univ. of California, Riverside, CA (United States)
  2. Univ. of California, Riverside, CA (United States); Claremont Colleges (United States)
  3. Univ. of Toledo, OH (United States)
  4. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
  5. Florida State Univ., Tallahassee, FL (United States)
  6. Bruker Biospin Corporation, Billerica, MA (United States)
  7. Bruker Switzerland AG, Fallanden (Switzerland)
  8. Univ. of Florida, Gainesville, FL (United States)
+ Show Author Affiliations

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate C$$\bar{β}$$ and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

Research Organization:
Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
Sponsoring Organization:
USDOE: National Science Foundation (NSF); National Institutes of Health (NIH)
Grant/Contract Number:
AC05-76RL01830
OSTI ID:
1845011
Report Number(s):
PNNL-SA-165557
Journal Information:
Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 2 Vol. 119; ISSN 0027-8424
Publisher:
National Academy of SciencesCopyright Statement
Country of Publication:
United States
Language:
English

References (77)

Solution‐State 17 O Quadrupole Central‐Transition NMR Spectroscopy in the Active Site of Tryptophan Synthase journal December 2015
Characterization of PEGylated Asparaginase: New Opportunities from NMR Analysis of Large PEGylated Therapeutics journal January 2019
UCSF Chimera?A visualization system for exploratory research and analysis journal January 2004
Converging nuclear magnetic shielding calculations with respect to basis and system size in protein systems journal May 2015
AFNMR: automated fragmentation quantum mechanical calculation of NMR chemical shifts for biomolecules journal August 2015
Rotational-echo double-resonance NMR journal January 1989
Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex journal March 2012
31P NMR spectroscopy senses the microenvironment of the 5′-phosphate group of enzyme-bound pyridoxal 5′-phosphate journal November 2011
Controlling reaction specificity in pyridoxal phosphate enzymes journal November 2011
Catalytic roles of βLys87 in tryptophan synthase: 15N solid state NMR studies journal September 2015
Visualizing the tunnel in tryptophan synthase with crystallography: Insights into a selective filter for accommodating indole and rejecting water journal March 2016
Broadband homonuclear correlation spectroscopy driven by combined R2nv sequences under fast magic angle spinning for NMR structural analysis of organic and biological solids journal July 2013
NMR spectroscopy up to 35.2 T using a series-connected hybrid magnet journal November 2017
Sensitivity boosts by the CPMAS CryoProbe for challenging biological assemblies journal February 2020
Chemical shift tensors: Theory and application to molecular structural problems journal May 2011
NMR crystallography of molecular organics journal June 2020
NMR crystallography: Applications to inorganic materials journal October 2014
Direct dynamic nuclear polarization of 15N and 13C spins at 14.1 T using a trityl radical and magic angle spinning journal August 2019
Structure of the 30 kDa HIV-1 RNA Dimerization Signal by a Hybrid Cryo-EM, NMR, and Molecular Dynamics Approach journal March 2018
Tryptophan synthase: the workings of a channeling nanomachine journal June 2008
CryoEM Structure Refinement by Integrating NMR Chemical Shifts with Molecular Dynamics Simulations journal January 2017
Substrate Binding Induces Conformational Changes in a Class A β-lactamase That Prime It for Catalysis journal January 2018
NMR Crystallography of Enzyme Active Sites: Probing Chemically Detailed, Three-Dimensional Structure in Tryptophan Synthase journal March 2013
Substitution of glutamic acid 109 by aspartic acid alters the substrate specificity and catalytic activity of the .beta.-subunit in the tryptophan synthase bienzyme complex from Salmonella typhimurium journal February 1992
Stereochemistry and mechanism of a new single-turnover, half-transamination reaction catalyzed by the tryptophan synthase .alpha.2.beta.2 complex journal January 1987
Microcrystals of tryptophan synthase .alpha.2.beta.2 complex from Salmonella typhimurium are catalytically active journal August 1987
Inhibition of alanine racemase by alanine phosphonate: detection of an imine linkage to pyridoxal 5'-phosphate in the enzyme-inhibitor complex by solid-state nitrogen-15 nuclear magnetic resonance journal July 1988
Stereoelectronic control of bond formation in Escherichia coli tryptophan synthase: substrate specificity and enzymatic synthesis of the novel amino acid dihydroisotryptophan journal September 1988
The tryptophan synthase bienzyme complex transfers indole between the .alpha.- and .beta.-sites via a 25-30 .ANG. long tunnel journal September 1990
Synergistic Effects on Escape of a Ligand from the Closed Tryptophan Synthase Bienzyme Complex journal December 2005
Allostery and Substrate Channeling in the Tryptophan Synthase Bienzyme Complex: Evidence for Two Subunit Conformations and Four Quaternary States journal September 2013
Allosteric Regulation of Substrate Channeling in Tryptophan Synthase:  Modulation of the l -Serine Reaction in Stage I of the β-Reaction by α-Site Ligands † , ‡ journal July 2007
QM/MM Study of the NMR Spectroscopy of the Retinyl Chromophore in Visual Rhodopsin journal June 2005
Toward the Quantum Chemical Calculation of NMR Chemical Shifts of Proteins. 3. Conformational Sampling and Explicit Solvents Model journal October 2012
Observation of an aminoacrylate enzyme intermediate in the tryptophan synthase reaction by solid-state NMR journal December 1995
Oxygen-17 nuclear magnetic resonance-spectroscopic studies of carbonmonoxy hemoproteins journal March 1989
X-ray and NMR Crystallography in an Enzyme Active Site: The Indoline Quinonoid Intermediate in Tryptophan Synthase journal January 2011
Quadrupole Central Transition 17 O NMR Spectroscopy of Biological Macromolecules in Aqueous Solution journal December 2010
Solid-State NMR Crystallography through Paramagnetic Restraints journal March 2012
A General Protocol for Determining the Structures of Molecularly Ordered but Noncrystalline Silicate Frameworks journal April 2013
De Novo Determination of the Crystal Structure of a Large Drug Molecule by Crystal Structure Prediction-Based Powder NMR Crystallography journal November 2013
NMR Studies of Protonation and Hydrogen Bond States of Internal Aldimines of Pyridoxal 5′-Phosphate Acid–Base in Alanine Racemase, Aspartate Aminotransferase, and Poly- l -lysine journal November 2013
Protonation States of the Tryptophan Synthase Internal Aldimine Active Site from Solid-State NMR Spectroscopy: Direct Observation of the Protonated Schiff Base Linkage to Pyridoxal-5′-Phosphate journal September 2014
Picometer Resolution Structure of the Coordination Sphere in the Metal-Binding Site in a Metalloprotein by NMR journal September 2020
Improved Accuracy from Joint X-ray and NMR Refinement of a Protein–RNA Complex Structure journal January 2016
HIV-1 Capsid Function Is Regulated by Dynamics: Quantitative Atomic-Resolution Insights by Integrating Magic-Angle-Spinning NMR, QM/MM, and MD journal October 2016
NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity journal November 2016
Positional Variance in NMR Crystallography journal February 2017
Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble journal April 2018
Deciphering the Allosterically Driven Conformational Ensemble in Tryptophan Synthase Evolution journal July 2019
Fast Microsecond Dynamics of the Protein–Water Network in the Active Site of Human Carbonic Anhydrase II Studied by Solid-State NMR Spectroscopy journal October 2019
Molecular Properties from Combined QM/MM Methods. 2. Chemical Shifts in Large Molecules journal March 2000
Determination of molecular symmetry in crystalline naphthalene using solid-state NMR journal September 1993
High-resolution structure of the Shigella type-III secretion needle by solid-state NMR and cryo-electron microscopy journal September 2014
Direct visualization of critical hydrogen atoms in a pyridoxal 5′-phosphate enzyme journal October 2017
Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex journal June 2019
Structure determination of an amorphous drug through large-scale NMR predictions journal May 2021
Structural basis for chain release from the enacyloxin polyketide synthase journal September 2019
Atomic-resolution protein structure determination by cryo-EM journal October 2020
A genomic overview of pyridoxal-phosphate-dependent enzymes journal September 2003
Benchmark fragment-based 1 H, 13 C, 15 N and 17 O chemical shift predictions in molecular crystals journal January 2016
Predicting anisotropic thermal displacements for hydrogens from solid-state NMR: a study on hydrogen bonding in polymorphs of palmitic acid journal January 2018
A Bayesian approach to NMR crystal structure determination journal January 2019
Accurate and cost-effective NMR chemical shift predictions for proteins using a molecules-in-molecules fragmentation-based method journal January 2020
Solid-state NMR approaches to investigate large enzymes in complex with substrates and inhibitors journal December 2020
Ab initio calculations of NMR chemical shifts journal February 2008
Sideband intensities in NMR spectra of samples spinning at the magic angle journal December 1980
Joint neutron crystallographic and NMR solution studies of Tyr residue ionization and hydrogen bonding: Implications for enzyme-mediated proton transfer journal April 2015
Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy journal October 2015
A structural model for Alzheimer's  -amyloid fibrils based on experimental constraints from solid state NMR journal December 2002
Conformation and reaction specificity in pyridoxal phosphate enzymes. journal April 1966
Pyridoxal Enzymes: Mechanistic Diversity and Uniformity journal September 1995
NMR crystallography: structure and properties of materials from solid-state nuclear magnetic resonance observables journal May 2017
Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors journal September 2019
Secondary and Tertiary Structural Effects on Protein NMR Chemical Shifts: an ab Initio Approach journal June 1993
Fibril structure of amyloid-β(1–42) by cryo–electron microscopy journal September 2017
Pyridoxal 5′-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase journal July 2012

Similar Records

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
NMR-assisted crystallography
integrated structural biology
pyridoxal-5"-phosphate
solid-state NMR
tryptophan synthase