Matthew P. Blakeley

Metal Ion Roles and the Movement of Hydrogen during Reaction Catalyzed by D-Xylose Isomerase: A Joint X-Ray and Neutron Diffraction Study

Conversion of aldo to keto sugars by the metalloenzyme D-xylose isomerase (XI) is a multistep reaction that involves hydrogen transfer. We have determined the structure of this enzyme by neutron diffraction in order to locate H atoms (or their isotope D). Two studies are presented, one of XI containing cadmium and cyclic D-glucose (before sugar ring opening has occurred), and the other containing nickel and linear D-glucose (after ring opening has occurred but before isomerization). Previously we reported the neutron structures of ligand-free enzyme and enzyme with bound product. The data show that His54 is doubly protonated on the ring N in all four structures. Lys289 is neutral before ring opening and gains a proton after this; the catalytic metal-bound water is deprotonated to hydroxyl during isomerization and O5 is deprotonated. These results lead to new suggestions as to how changes might take place over the course of the reaction.

Neutron crystallography: opportunities, challenges, and limitations.

Neutron crystallography has had an important, but relatively small role in structural biology over the years. In this review of recently determined neutron structures, a theme emerges of a field currently expanding beyond its traditional boundaries, to address larger and more complex problems, with smaller samples and shorter data collection times, and employing more sophisticated structure determination and refinement methods. The origin of this transformation can be found in a number of advances including first, the development of neutron image-plates and quasi-Laue methods at nuclear reactor neutron sources and the development of time-of-flight Laue methods and electronic detectors at spallation neutron sources; second, new facilities and methods for sample perdeuteration and crystallization; third, new approaches and computational tools for structure determination.

Neutron cryo-crystallography captures the protonation state of ferryl heme in a peroxidase

Peroxidase proton placement Heme enzymes catalyze a variety of biochemical oxidations through the activation of oxygen by iron. Casadei et al. used neutron crystallography to elucidate the mechanism of cytochrome c peroxidase (see the perspective by Groves and Boaz). In the highly reactive intermediate state termed compound I, the iron(IV) oxo, or ferryl, fragment was not protonated, whereas a nearby histidine residue was protonated. The sensitivity of neutron scattering to proton locations revealed these protonation states, where more common techniques, such as x-ray diffraction, have yielded more ambiguous results. Science, this issue p. 193; see also p. 142 The sensitivity of neutron scattering to proton locations clarifies the acid/base chemistry of a biochemical oxidation. [Also see Perspective by Groves and Boaz] Heme enzymes activate oxygen through formation of transient iron-oxo (ferryl) intermediates of the heme iron. A long-standing question has been the nature of the iron-oxygen bond and, in particular, the protonation state. We present neutron structures of the ferric derivative of cytochrome c peroxidase and its ferryl intermediate; these allow direct visualization of protonation states. We demonstrate that the ferryl heme is an Fe(IV)=O species and is not protonated. Comparison of the structures shows that the distal histidine becomes protonated on formation of the ferryl intermediate, which has implications for the understanding of O–O bond cleavage in heme enzymes. The structures highlight the advantages of neutron cryo-crystallography in probing reaction mechanisms and visualizing protonation states in enzyme intermediates.

Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase

We present results of combined studies of the enzyme human aldose reductase (h-AR, 36 kDa) using single-crystal x-ray data (0.66 Å, 100K; 0.80 Å, 15K; 1.75 Å, 293K), neutron Laue data (2.2 Å, 293K), and quantum mechanical modeling. These complementary techniques unveil the internal organization and mobility of the hydrogen bond network that defines the properties of the catalytic engine, explaining how this promiscuous enzyme overcomes the simultaneous requirements of efficiency and promiscuity offering a general mechanistic view for this class of enzymes.

Neutron macromolecular crystallography with LADI‐III

At the Institut Laue-Langevin, a new neutron Laue diffractometer LADI-III has been fully operational since March 2007. LADI-III is dedicated to neutron macromolecular crystallography at medium to high resolution (2.5-1.5 Å) and is used to study key H atoms and water structure in macromolecular structures. An improved detector design and readout system has been incorporated so that a miniaturized reading head located inside the drum scans the image plate. From comparisons of neutron detection efficiency (DQE) with the original LADI-I instrument, the internal transfer of the image plates and readout system provides an approximately threefold gain in neutron detection. The improved performance of LADI-III, coupled with the use of perdeuterated biological samples, now allows the study of biological systems with crystal volumes of 0.1-0.2 mm(3), as illustrated here by the recent studies of type III antifreeze protein (AFP; 7 kDa). As the major bottleneck for neutron macromolecular studies has been the large crystal volumes required, these recent developments have led to an expansion of the field, extending the size and the complexity of the systems that can be studied and reducing the data-collection times required.

High-resolution neutron protein crystallography with radically small crystal volumes: application of perdeuteration to human aldose reductase.

Neutron diffraction data have been collected to 2.2 Angstrom resolution from a small (0.15 mm(3)) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase [h-AR(D)], subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm(3) are reported. Neutron data were recorded to 2 Angstrom resolution, with subsequent data analysis using data to 2.2 Angstrom. This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples.

Joint X-ray/Neutron Crystallographic Study of HIV-1 Protease with Clinical Inhibitor Amprenavir: Insights for Drug Design

HIV-1 protease is an important target for the development of antiviral inhibitors to treat AIDS. A room-temperature joint X-ray/neutron structure of the protease in complex with clinical drug amprenavir has been determined at 2.0 Å resolution. The structure provides direct determination of hydrogen atom positions in the enzyme active site. Analysis of the enzyme-drug interactions suggests that some hydrogen bonds may be weaker than deduced from the non-hydrogen interatomic distances. This information may be valuable for the design of improved protease inhibitors.

The active site protonation states of perdeuterated Toho‐1 β‐lactamase determined by neutron diffraction support a role for Glu166 as the general base in acylation

Room temperature neutron diffraction data of the fully perdeuterated Toho‐1 R274N/R276N double mutant β‐lactamase in the apo form were used to visualize deuterium atoms within the active site of the enzyme. This perdeuterated neutron structure of the Toho‐1 R274N/R276N reveals the clearest picture yet of the ground‐state active site protonation states and the complete hydrogen‐bonding network in a β‐lactamase enzyme. The ground‐state active site protonation states detailed in this neutron diffraction study are consistent with previous high‐resolution X‐ray studies that support the role of Glu166 as the general base during the acylation reaction in the class A β‐lactamase reaction pathway.

Neutron Diffraction Studies of a Class a Beta-Lactamase Toho-1 E166A R274N R276N Triple Mutant

beta-Lactam antibiotics have been used effectively over several decades against many types of bacterial infectious diseases. However, the most common cause of resistance to the beta-lactam antibiotics is the production of beta-lactamase enzymes that inactivate beta-lactams by rapidly hydrolyzing the amide group of the beta-lactam ring. Specifically, the class A extended-spectrum beta-lactamases (ESBLs) and inhibitor-resistant enzymes arose that were capable of hydrolyzing penicillins and the expanded-spectrum cephalosporins and monobactams in resistant bacteria, which lead to treatment problems in many clinical settings. A more complete understanding of the mechanism of catalysis of these ESBL enzymes will impact current antibiotic drug discovery efforts. Here, we describe the neutron structure of the class A, CTX-M-type ESBL Toho-1 E166A/R274N/R276N triple mutant in its apo form, which is the first reported neutron structure of a beta-lactamase enzyme. This neutron structure clearly reveals the active-site protonation states and hydrogen-bonding network of the apo Toho-1 ESBL prior to substrate binding and subsequent acylation. The protonation states of the active-site residues Ser70, Lys73, Ser130, and Lys234 in this neutron structure are consistent with the prediction of a proton transfer pathway from Lys73 to Ser130 that is likely dependent on the conformation of Lys73, which has been hypothesized to be coupled to the protonation state of Glu166 during the acylation reaction. Thus, this neutron structure is in agreement with a proposed mechanism for acylation that identifies Glu166 as the general base for catalysis.

Unambiguous determination of H-atom positions: comparing results from neutron and high-resolution X-ray crystallography.

The locations of H atoms in biological structures can be difficult to determine using X-ray diffraction methods. Neutron diffraction offers a relatively greater scattering magnitude from H and D atoms. Here, 1.65 A resolution neutron diffraction studies of fully perdeuterated and selectively CH(3)-protonated perdeuterated crystals of Pyrococcus furiosus rubredoxin (D-rubredoxin and HD-rubredoxin, respectively) at room temperature (RT) are described, as well as 1.1 A resolution X-ray diffraction studies of the same protein at both RT and 100 K. The two techniques are quantitatively compared in terms of their power to directly provide atomic positions for D atoms and analyze the role played by atomic thermal motion by computing the sigma level at the D-atom coordinate in simulated-annealing composite D-OMIT maps. It is shown that 1.65 A resolution RT neutron data for perdeuterated rubredoxin are approximately 8 times more likely overall to provide high-confidence positions for D atoms than 1.1 A resolution X-ray data at 100 K or RT. At or above the 1.0sigma level, the joint X-ray/neutron (XN) structures define 342/378 (90%) and 291/365 (80%) of the D-atom positions for D-rubredoxin and HD-rubredoxin, respectively. The X-ray-only 1.1 A resolution 100 K structures determine only 19/388 (5%) and 8/388 (2%) of the D-atom positions above the 1.0sigma level for D-rubredoxin and HD-rubredoxin, respectively. Furthermore, the improved model obtained from joint XN refinement yielded improved electron-density maps, permitting the location of more D atoms than electron-density maps from models refined against X-ray data only.