Yael S. Balazs

In situ molecular NMR picture of bioavailable calcium stabilized as amorphous CaCO3 biomineral in crayfish gastroliths

Bioavailable calcium is maintained by some crustaceans, in particular freshwater crayfish, by stabilizing amorphous calcium carbonate (ACC) within reservoir organs—gastroliths, readily providing the Ca2+ needed to build a new exoskeleton. Despite the key scientific and biomedical importance of the in situ molecular-level picture of biogenic ACC and its stabilization in a bioavailable form, its description has eluded efforts to date. Herein, using multinuclear NMR, we accomplish in situ molecular-level characterization of ACC within intact gastroliths of the crayfish Cherax quadricarinatus. In addition to the known CaCO3, chitin scaffold and inorganic phosphate (Pi), we identify within the gastrolith two primary metabolites, citrate and phosphoenolpyruvate (PEP) and quantify their abundance by applying solution NMR techniques to the gastrolith “soluble matrix.” The long-standing question on the physico-chemical state of ACC stabilizing, P-bearing moieties within the gastrolith is answered directly by the application of solid state rotational-echo double-resonance (REDOR) and transferred-echo double-resonance (TEDOR) NMR to the intact gastroliths: Pi and PEP are found molecularly dispersed throughout the ACC as a solid solution. Citrate carboxylates are found < 5 Å from a phosphate (intermolecular C⋯P distance), an interaction that must be mediated by Ca2+. The high abundance and extensive interactions of these molecules with the ACC matrix identify them as the central constituents stabilizing the bioavailable form of calcium. This study further emphasizes that it is imperative to characterize the intact biogenic CaCO3. Solid state NMR spectroscopy is shown to be a robust and accessible means of determining composition, internal structure, and molecular functionality in situ.

Glycosynthase activity of Geobacillus stearothermophilus GH52 β-xylosidase : Efficient synthesis of xylooligosaccharides from α-D-xylopyranosyl fluoride through a conjugated reaction

Glycosynthases are mutant glycosidases in which the acidic nucleophile is replaced by a small inert residue. In the presence of glycosyl fluorides of the opposite anomeric configuration (to that of their natural substrates), these enzymes can catalyze glycosidic bond formation with various acceptors. In this study we demonstrate that XynB2E335G, a nucleophile‐deficient mutant of a glycoside hydrolase family 52 β‐xylosidase from G. stearothermophilus, can function as an efficient glycosynthase, using α‐D‐xylopyranosyl fluoride as a donor and various aryl sugars as acceptors. The mutant enzyme can also catalyze the self‐condensation reaction of α‐D‐xylopyranosyl fluoride, providing mainly α‐D‐xylobiosyl fluoride. The self‐condensation kinetics exhibited apparent classical Michaelis–Menten behavior, with kinetic constants of 1.3 s−1 and 2.2 mM for kcat and KM(acceptor), respectively, and a kcat/KM(acceptor) value of 0.59 s−1 mM−1. When the β‐xylosidase E335G mutant was combined with a glycoside hydrolase family 10 glycosynthase, high‐molecular‐weight xylooligomers were readily obtained from the affordable α‐D‐xylopyranosyl fluoride as the sole substrate.

A New Family of Carbohydrate Esterases Is Represented by a GDSL Hydrolase/Acetylxylan Esterase from Geobacillus stearothermophilus

Background: Acetylxylan esterases are enzymes that remove acetyl groups from the hemicellulolytic polymer xylan. Results: The axe2 gene product in Geobacillus stearothermophilus removes acetyl groups from acetylated xylan and xylosaccharides. Conclusion: Axe2 represents a new serine carbohydrate esterase family. Significance: The findings may provide new routes for the efficient utilization of biomass as a renewable energy source. Acetylxylan esterases hydrolyze the ester linkages of acetyl groups at positions 2 and/or 3 of the xylose moieties in xylan and play an important role in enhancing the accessibility of xylanases to the xylan backbone. The hemicellulolytic system of the thermophilic bacterium Geobacillus stearothermophilus T-6 comprises a putative acetylxylan esterase gene, axe2. The gene product belongs to the GDSL hydrolase family and does not share sequence homology with any of the carbohydrate esterases in the CAZy Database. The axe2 gene is induced by xylose, and the purified gene product completely deacetylates xylobiose peracetate (fully acetylated) and hydrolyzes the synthetic substrates 2-naphthyl acetate, 4-nitrophenyl acetate, 4-methylumbelliferyl acetate, and phenyl acetate. The pH profiles for kcat and kcat/Km suggest the existence of two ionizable groups affecting the binding of the substrate to the enzyme. Using NMR spectroscopy, the regioselectivity of Axe2 was directly determined with the aid of one-dimensional selective total correlation spectroscopy. Methyl 2,3,4-tri-O-acetyl-β-d-xylopyranoside was rapidly deacetylated at position 2 or at positions 3 and 4 to give either diacetyl or monoacetyl intermediates, respectively; methyl 2,3,4,6-tetra-O-acetyl-β-d-glucopyranoside was initially deacetylated at position 6. In both cases, the complete hydrolysis of the intermediates occurred at a much slower rate, suggesting that the preferred substrate is the peracetate sugar form. Site-directed mutagenesis of Ser-15, His-194, and Asp-191 resulted in complete inactivation of the enzyme, consistent with their role as the catalytic triad. Overall, our results show that Axe2 is a serine acetylxylan esterase representing a new carbohydrate esterase family.

Molecular Level Characterization of the Inorganic−Bioorganic Interface by Solid State NMR: Alanine on a Silica Surface, a Case Study

The molecular interface between bioorganics and inorganics plays a key role in diverse scientific and technological research areas including nanoelectronics, biomimetics, biomineralization, and medical applications such as drug delivery systems and implant coatings. However, the physical/chemical basis of recognition of inorganic surfaces by biomolecules remains unclear. The molecular level elucidation of specific interfacial interactions and the structural and dynamical state of the surface bound molecules is of prime scientific importance. In this study, we demonstrate the ability of solid state NMR methods to accomplish these goals. L-[1-(13)C,(15)N]Alanine loaded onto SBA-15 mesoporous silica with a high surface area served as a model system. The interacting alanine moiety was identified as the -NH(3)(+) functional group by (15)N{(1)H}SLF NMR. (29)Si{(15)N} and (15)N{(29)Si}REDOR NMR revealed intermolecular interactions between the alanine -NH(3)(+) and three to four surface Si species, predominantly Q(3), with similar internuclear N...Si distances of 4.0-4.2 A. Distinct dynamic states of the adsorbed biomolecules were identified by (15)N{(13)C}REDOR NMR, indicating both bound and free alanine populations, depending on hydration level and temperature. In the bound populations, the -NH(3)(+) group is surface anchored while the free carboxylate end undergoes librations, implying the carboxylate has small or no contributions to surface binding. When surface water clusters grow bigger with increased hydration, the libration amplitude of the carboxyl end amplifies, until onset of dissolution occurs. Our measurements provide the first direct, comprehensive, molecular-level identification of the bioorganic-inorganic interface, showing binding functional groups, geometric constraints, stoichiometry, and dynamics, both for the adsorbed amino acid and the silica surface.

Synthesis and properties of a corrole with small and electron-withdrawing substitutents, 5,15-bis(trifluoromethyl)-10-pentafluorophenylcorrole

Several methodologies for the synthesis of corroles that carry minimal-sized electron-withdrawing substituents at the meso-C atoms were investigated; of these the one based on dypyrromethane/aldehyde condensation was fruitful. The new corrole with one C6F5 and two CF3 groups, as well as its cobalt(III) complex, were fully characterized by spectroscopy and X-ray crystallography.

Identifying critical unrecognized sugar–protein interactions in GH10 xylanases from Geobacillus stearothermophilus using STD NMR

1H solution NMR spectroscopy is used synergistically with 3D crystallographic structures to map experimentally significant hydrophobic interactions upon substrate binding in solution under thermodynamic equilibrium. Using saturation transfer difference spectroscopy (STD NMR), a comparison is made between wild‐type xylanase XT6 and its acid/base catalytic mutant E159Q – a non‐active, single‐heteroatom alteration that has been previously utilized to measure binding thermodynamics across a series of xylooligosaccharide–xylanase complexes [Zolotnitsky et al. (2004) Proc Natl Acad Sci USA 101, 11275–11280). In this study, performing STD NMR of one substrate screens binding interactions to two proteins, avoiding many disadvantages inherent to the technique and clearly revealing subtle changes in binding induced upon mutation of the catalytic Glu. To visualize and compare the binding epitopes of xylobiose–xylanase complexes, a ‘SASSY’ plot (saturation difference transfer spectroscopy) is used. Two extraordinarily strong, but previously unrecognized, non‐covalent interactions with H2–5 of xylobiose were observed in the wild‐type enzyme but not in the E159Q mutant. Based on the crystal structure, these interactions were assigned to tryptophan residues at the −1 subsite. The mutant selectively binds only the β–xylobiose anomer. The 1H solution NMR spectrum of a xylotriose–E159Q complex displays non‐uniform broadening of the NMR signals. Differential broadening provides a unique subsite assignment tool based on structural knowledge of face‐to‐face stacking with a conserved tyrosine residue at the +1 subsite. The results obtained herein by substrate‐observed NMR spectroscopy are discussed further in terms of methodological contributions and mechanistic understanding of substrate‐binding adjustments upon a charge change in the E159Q construct.

A paradigm for solvent and temperature induced conformational changes.

Knowing the chemical formula of small molecules, which are the basic building blocks of organic and inorganic materials, is not always enough to predict their behavior because also the environment can have a significant influence. Distinct molecular conformations can arise from free rotations around the bonds resulting in a number of potential threedimensional conformations (stereoisomers) and differences in activities can be induced, despite the fact that the atomic composition of the original molecule is unchanged and the number of single bonds conserved. Examples include the surprising results that biological enzymes in neat organic solvents can show increased stability and profound changes in selectivity compared to aqueous environments. The stabilization or recognition of a single molecular conformation over others is necessary in divers phenomena, such as: bioseparations, the ability of peptides to pass membranes, and many protein behaviors such as: translocation, folding, and substrate or inhibitor binding. Herein we examine a small organic molecule, 4-[(tert-butoxycarbonyl)amino]-2-hydroxybutanoic acid (Boc-amine; Figure 1) that exhibits multiple conformations. The conformations observed depend on solvent and temperature. Bocamine is explored via X-ray, NMR, membrane–activity assays, and DFT calculations. In addition, its behaviors are contrasted with those of other small isomeric organic molecules, including three Boc-amine analogues (Figure 2) with the ultimate goal of understanding the physical and chemical details of how the environment induces the observed conformational isomerism. The crystal structure can be described by two different chains (Figure 1). Each individual chain contains conformationally identical molecules (“open” and “closed”) hydrogen bonded via their hydroxyl and carboxylate groups and extending by the screw axis along b (Figure 1 a). No solvent molecules are present in the crystal structure.