The Catalytic Role of Glutamate 151 in the Leucine Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e

Glutamate 151 has been proposed to act as the general acid/base during the peptide hydrolysis reaction catalyzed by the co-catalytic metallohydrolase from Aeromonas proteolytica (AAP). However, to date, no direct evidence has been reported for the role of Glu-151 during catalytic turnover by AAP. In order to elucidate the catalytic role of Glu-151, altered AAP enzymes have been prepared in which Glu-151 has been substituted with a glutamine, an alanine, and an aspartate. The Michaelis constant (Km) does not change upon substitution to aspartate or glutamine, but the rate of the reaction changes drastically in the following order: glutamate (100% activity), aspartate (0.05%), glutamine (0.004%), and alanine (0%). Examination of the pH dependence of the kinetic constants kcat and Km revealed a change in the pKa of a group that ionizes at pH 4.8 in recombinant leucine aminopeptidase (rAAP) to 4.2 for E151D-AAP. The remaining pKa values at 5.2, 7.5, and 9.9 do not change. Proton inventory studies indicate that one proton is transferred in the rate-limiting step of the reaction at pH 10.50 for both rAAP and E151D-AAP, but at pH 6.50 two protons and general solvation effects are responsible for the observed effects in the reaction catalyzed by rAAP and E151D-AAP, respectively. Based on these data, Glu-151 is intrinsically involved in the peptide hydrolysis reaction catalyzed by AAP and can be assigned the role of a general acid and base

Determination of local material properties of OSB sample by coupling advanced imaging techniques and morphology-based FEM simulation

This is the publisher’s final pdf. The published article is copyrighted by Walter de Gruyter & Co. and can be found at: http://www.degruyter.com/.The goal was to determine local mechanical properties inside of oriented strand board (OSB) based on a realistic morphology-based finite element (FE) model and data acquired from a physical test performed on the same material. The spatial information and local grayscale intensity from CT-scans obtained from small OSB sample was transformed into a 2D regular morphology-based FE mesh with corresponding material properties. The model was then used to simulate the actual compression test performed on the specimen using simplified boundary conditions. The simulated strain fields from the model were compared with the actual strain field measured on the specimen surface during the compression test by means of a full-field optical method, named digital image correlation (DIC). Finally, the original set of material properties was adjusted by an iterative procedure to minimize the difference between the simulated and the measured strain data. The results show that the developed procedure is useful to find local material properties as well as for morphological modeling without the need of segmentation of the image data. The achieved results serve as a prerequisite for full 3D analyses of the complex materials

Lysine Biosynthesis in Bacteria: A Metallodesuccinylase as a Potential Antimicrobial Target

In this review, we summarize the recent literature on dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) enzymes, with an emphasis on structure–function studies that provide insight into the catalytic mechanism. Crystallographic data have also provided insight into residues that might be involved in substrate and hence inhibitor recognition and binding. These data have led to the design and synthesis of several new DapE inhibitors, which are described along with what is known about how inhibitors interact with the active site of DapE enzymes, including the efficacy of a moderately strong DapE inhibitor

An Analytical Method for Detecting Toxic Metal Cations Using Cyclotriveratrylene Derivative Capped Gold Nanoparticles

Cyclotriveratrylene-oxime (CTV-oxime) derivatives that terminate with a dithiolate linker were synthesized enabling the supramolecular scaffold to adhere to gold nanoparticles (AuNPs) with the bowl-shaped cavity of the CTV scaffold exposed for utilization in host–guest chemistry. Exposure of these CTV functionalized AuNPs to varying concentrations of di- and trivalent metal cations resulted in the formation of large CTV-AuNP polymeric clusters and an accompanying a shift in the plasmon resonance. These interactions between the CTV-AuNPs and the metal cations in solution provides proof-of-concept that supramolecular functionalized AuNPs can be used as a simple and straightforward, on-site detection system for toxic metal cations in solution. The order of binding affinity of the metals studied based on observed Kd values is Cu2+ \u3e Zn2+ \u3e Pb2+ \u3e Hg2+ \u3e Eu3+ \u3e Cd2+

Co-Catalytic Metallopeptidases as Pharmaceutical Targets

Understanding the reaction mechanism of co-catalytic metallopeptidases provides a starting point for the design and synthesis of new molecules that can be screened as potential pharmaceuticals. Many of the enzymes that contain co-catalytic metallo-active sites play important roles in cellular processes such as tissue repair, protein maturation, hormone level regulation, cell-cycle control and protein degradation. Therefore, these enzymes play central roles in several disease states including cancer, HIV, stroke, diabetes, bacterial infections, neurological processes, schizophrenia, seizure disorders, and amyotrophic lateral sclerosis. The mechanism of AAP, an aminopeptidase from Aeromonas proteolytica, is one of the best-characterized examples of a metallopeptidase containing a co-catalytic metallo-active site, although this enzyme is not a specific pharmaceutical target at this time. As a large majority of co-catalytic metallopeptidases contain active sites that are nearly identical to the one observed in AAP, the major steps of their catalytic mechanisms are likely to be very similar. With this in mind, it is possible to propose a general catalytic mechanism for the hydrolysis of amino acid substrates

Inhibitors of Bacterial \u3cem\u3eN\u3c/em\u3e-succinyl-L,L-diaminopimelic Acid Desuccinylase (DapE) and Demonstration of in vitro Antimicrobial Activity

The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) is a critical bacterial enzyme for the construction of the bacterial cell wall. A screen biased toward compounds containing zinc-binding groups (ZBG’s) including thiols, carboxylic acids, boronic acids, phosphonates and hydroxamates has delivered a number of micromolar inhibitors of DapE from Haemophilus influenzae, including the low micromolar inhibitor L-captopril (IC50 = 3.3 μM, Ki = 1.8 μM). In vitro antimicrobial activity was demonstrated for l-captopril against Escherichia coli

Direct Patterning of a Cyclotriveratrylene Derivative for Directed Self-assembly of C60

A novel apex-modified cyclotriveratrylene (CTV) derivative with an attached thiolane-containing lipoic acid linker was directly patterned onto gold substrates via dip-pen nanolithography (DPN). The addition of a dithiolane-containing linker to the apex of CTV provides a molecule that can adhere to a gold surface with its bowl-shaped cavity directed away from the surface, thereby providing a surface-bound CTV host that can be used for the directed assembly of guest molecules. Subsequent exposure of these CTV microarrays to C60 in toluene resulted in the directed assembly of predesigned, spatially controlled, high-density microarrays of C60. The molecular recognition capabilities of this CTV template toward C60 provides proof-of-concept that supramolecular CTV scaffolds can be directly patterned onto surfaces providing a foundation for the development of organic electronic and optoelectronic materials

Structural Basis for Catalysis by the Mono- and Dimetalated Forms of the \u3cem\u3edapE\u3c/em\u3e-Encoded \u3cem\u3eN\u3c/em\u3e-succinyl-L,L-Diaminopimelic Acid Desuccinylase

Biosynthesis of lysine and meso-diaminopimelic acid in bacteria provides essential components for protein synthesis and construction of the bacterial peptidoglycan cell wall. The dapE operon enzymes synthesize both meso-diaminopimelic acid and lysine and, therefore, represent potential targets for novel antibacterials. The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase functions in a late step of the pathway and converts N-succinyl-l,l-diaminopimelic acid to l,l-diaminopimelic acid and succinate. Deletion of the dapE gene is lethal to Helicobacter pylori and Mycobacterium smegmatis, indicating that DapE\u27s are essential for cell growth and proliferation. Since there are no similar pathways in humans, inhibitors that target DapE may have selective toxicity against only bacteria. A major limitation in developing antimicrobial agents that target DapE has been the lack of structural information. Herein, we report the high-resolution X-ray crystal structures of the DapE from Haemophilus influenzae with one and two zinc ions bound in the active site, respectively. These two forms show different activity. Based on these newly determined structures, we propose a revised catalytic mechanism of peptide bond cleavage by DapE enzymes. These structures provide important insight into catalytic mechanism of DapE enzymes as well as a structural foundation that is critical for the rational design of DapE inhibitors

Vibrational mechanics in an optical lattice: controlling transport via potential renormalization

We demonstrate theoretically and experimentally the phenomenon of vibrational resonance in a periodic potential, using cold atoms in an optical lattice as a model system. A high-frequency (HF) drive, with frequency much larger than any characteristic frequency of the system, is applied by phase-modulating one of the lattice beams. We show that the HF drive leads to the renormalization of the potential. We used transport measurements as a probe of the potential renormalization. The very same experiments also demonstrate that transport can be controlled by the HF drive via potential renormalization.Comment: Phys. Rev. Lett., in pres