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Manfred S Weiss

Publications and source records attributed to Manfred S Weiss.

27 records · Page 2Linked to original sources
PubMed2005

The role of Val-265 for flavin adenine dinucleotide (FAD) binding in pyruvate oxidase: FTIR, kinetic, and crystallographic studies on the enzyme variant V265A.

In pyruvate oxidase (POX) from Lactobacillus plantarum, valine 265 participates in binding the cofactor FAD and is responsible for the strained conformation of its isoalloxazine moiety that is visible in the crystal structure of POX. The contrasting effects of the conservative amino acid exchange V265A on the enzyme's catalytic properties, cofactor affinity, and protein structure were investigated. The most prominent effect of the exchange was observed in the 2.2 A crystal structure of the mutant POX. While the overall structures of the wild-type and the variant are similar, flavin binding in particular is clearly different. Local disorder at the isoalloxazine binding site prevents modeling of the complete FAD cofactor and two protein loops of the binding site. Only the ADP moiety shows well-defined electron density, indicating an "anchor" function for this part of the molecule. This notion is corroborated by competition experiments where ADP was used to displace FAD from the variant enzyme. Despite the fact that the affinity of FAD binding in the variant is reduced, the catalytic properties are very similar to the wild-type, and the redox potential of the bound flavin is the same for both proteins. The rate of electron transfer toward the flavin during turnover is reduced to one-third compared to the wild-type, but k(cat) remains unchanged. Redox-triggered FTIR difference spectroscopy of free FAD shows the nu(C(10a)=N(1)) band at 1548 cm(-)(1). In POX-V265A, this band is found at 1538 cm(-)(1) and thus shifted less strongly than in wild-type POX where it is found at 1534 cm(-)(1). Taking these observations together, the conservative exchange V265A in POX has a surprisingly small effect on the catalytic properties of the enzyme, whereas the effect on the three-dimensional structure is rather big.

Base Sequence↗
PubMed2005

Auto-rickshaw: an automated crystal structure determination platform as an efficient tool for the validation of an X-ray diffraction experiment.

The EMBL-Hamburg Automated Crystal Structure Determination Platform is a system that combines a number of existing macromolecular crystallographic computer programs and several decision-makers into a software pipeline for automated and efficient crystal structure determination. The pipeline can be invoked as soon as X-ray data from derivatized protein crystals have been collected and processed. It is controlled by a web-based graphical user interface for data and parameter input, and for monitoring the progress of structure determination. A large number of possible structure-solution paths are encoded in the system and the optimal path is selected by the decision-makers as the structure solution evolves. The processes have been optimized for speed so that the pipeline can be used effectively for validating the X-ray experiment at a synchrotron beamline.

Crystallography, X-Ray↗
PubMed2004

The high-resolution Structure of LeuB (Rv2995c) from Mycobacterium tuberculosis.

The crystal structure of the enzyme 3-isopropylmalate dehydrogenase (IPMDH) from Mycobacterium tuberculosis (LeuB, Mtb-IPMDH, Rv2995c) without substrate or co-factor was determined at 1.65 A resolution, which is the highest resolution reported for an IPMDH to date. The crystals contain two functional dimers in the asymmetric unit in an arrangement close to a tetramer of D2 symmetry. Despite the absence of a substrate or inhibitor bound to the protein, the structure of the monomer resembles the previously observed closed form of the enzyme more closely than the open form. A comparison with the substrate complex of IPMDH from Thiobacillus ferrooxidans and the co-factor complex of the Thermus thermophilus enzyme revealed a close relationship of the active-site architecture between the various bacterial enzymes. The inhibitor O-isobutenyl oxalylhydroxamate was found to bind to the active site of IPMDH in a mode similar to the substrate isopropylmalate.

3-Isopropylmalate Dehydrogenase↗
PubMed2004

Determination of a novel structure by a combination of long-wavelength sulfur phasing and radiation-damage-induced phasing.

The structure of the 115 amino-acid residue protein DsvC was determined based on the anomalous scattering provided by the five S atoms present in the structure. By collecting the diffraction data at a wavelength of 1.9 A, the anomalous signal provided by the S atoms was enhanced. However, significant radiation damage occurred during the course of the experiment, which led to differences between different parts of the data set. Only by dividing the total data set into five data sets was it possible to obtain phases; these could then be successfully extended to allow structure determination by the automated model-building program ARP/wARP. A computational correction for the radiation damage was found to significantly improve the success rate in determining the heavy-atom substructure and to improve phasing and refinement statistics.

Archaeal Proteins↗
PubMed2003

On the routine use of soft X-rays in macromolecular crystallography. Part II. Data-collection wavelength and scaling models.

Complete and highly redundant data sets were collected at nine different wavelengths between 0.80 and 2.65 A on a xenon derivative of porcine pancreatic elastase in both air and helium atmospheres. The magnitude of the anomalous signal, as assessed by the xenon-peak height in the anomalous difference Patterson synthesis, is affected by the wavelength of data collection as well as by the scaling model used. For data collected at wavelengths longer than 1.7 A, the use of a three-dimensional scaling protocol is essential in order to obtain the highest possible anomalous signal. Based on the scaling protocols currently available, the optimal wavelength range for data collection appears to be between 2.1 and 2.4 A. Beyond that, any further increase in signal will be compensated for or even superseded by a concomitant increase in noise, which cannot be fully corrected for. Data collection in a helium atmosphere yields higher I/sigma(I) values, but not significantly better anomalous differences, than data collection in air.

Air↗
PubMed2002

The active principle of garlic at atomic resolution.

Despite the fact that many cultures around the world value and utilize garlic as a fundamental component of their cuisine as well as of their medicine cabinets, relatively little is known about the plant's protein configuration that is responsible for the specific properties of garlic. Here, we report the three-dimensional structure of the garlic enzyme alliinase at 1.5 A resolution. Alliinase constitutes the major protein component in garlic bulbs, and it is able to cleave carbon-sulfur bonds. The active enzyme is a pyridoxal-5'-phosphate-dependent homodimeric glycoprotein and belongs to the class I family of pyridoxal-5'-phosphate-dependent enzymes. In addition, it contains a novel epidermal growth factor-like domain that makes it unique among all pyridoxal-5'-phosphate-dependent enzymes.

Binding Sites↗
PubMed2002

Metal binding to porcine pancreatic elastase: calcium or not calcium.

Porcine pancreatic elastase has been crystallized at slightly acidic pH under two similar but slightly different conditions. Diffraction data were collected at a wavelength of 1.5 A to a maximum resolution of 1.7 A. Both difference electron-density maps and anomalous difference electron-density maps suggest that in crystals grown from a sodium sulfate solution PPE binds Na(+) in its metal-binding site. In contrast, PPE binds Ca(2+) in crystals grown from a solution containing sodium citrate and calcium chloride. This observation is in contradiction to most PPE structures reported in the PDB. In addition to the metal-binding site, up to three other binding sites, which appear to be anion-binding sites, could be identified based on the observed anomalous intensity differences.

Animals↗
PubMed2002

Purification, characterization, and crystallization of alliinase from garlic.

Glycosylated dimeric alliinase (EC 4.4.1.4) was purified to homogeneity from its natural source, garlic. With 660 units/mg, the specific enzymatic activity of the pure enzyme is the highest reported to date. Based on both CD spectroscopy data and sequence-derived secondary structure prediction, the alpha-helix content of alliinase was estimated to be about 30%. Comparisons of all available amino acid sequences of alliinases revealed a common cysteine pattern of the type C-x18-19-C-x-C-x2-C-x5-C-x6-C in the N-terminal part of the sequences. This pattern is conserved in alliinases but absent in other pyridoxal 5'-phosphate-dependent enzymes. It suggests the presence of an epidermal growth factor-like domain in the three-dimensional structures of alliinases, making them unique among the various families of pyridoxal 5'-phosphate-dependent enzymes. Well-ordered three-dimensional crystals of garlic alliinase were obtained in four different forms. The best diffraction was observed with crystal form IV (space group P2(1)2(1)2(1), a=68.4, b=101.1, c=155.7 A) grown from an ammonium sulfate solution. These crystals diffract to at least 1.5 A resolution at a synchrotron source and are suitable for structure determination.

Amino Acid Sequence↗