Hans-Jürgen Hecht

Structure of the molybdopterin- bound Cnx1G domain links molybdenum and copper metabolism

The molybdenum cofactor is part of the active site of all molybdenum-dependent enzymes, except nitrogenase. The molybdenum cofactor consists of molybdopterin, a phosphorylated pyranopterin, with an ene-dithiolate coordinating molybdenum. The same pyranopterin-based cofactor is involved in metal coordination of the homologous tungsten-containing enzymes found in archea. The molybdenum cofactor is synthesized by a highly conserved biosynthetic pathway. In plants, the multidomain protein Cnx1 catalyses the insertion of molybdenum into molybdopterin. The Cnx1 G domain (Cnx1G), whose crystal structure has been determined in its apo form, binds molybdopterin with high affinity and participates in the catalysis of molybdenum insertion. Here we present two high-resolution crystal structures of Cnx1G in complex with molybdopterin and with adenylated molybdopterin (molybdopterin–AMP), a mechanistically important intermediate. Molybdopterin–AMP is the reaction product of Cnx1G and is subsequently processed in a magnesium-dependent reaction by the amino-terminal E domain of Cnx1 to yield active molybdenum cofactor. The unexpected identification of copper bound to the molybdopterin dithiolate sulphurs in both structures, coupled with the observed copper inhibition of Cnx1G activity, provides a molecular link between molybdenum and copper metabolism.

Effects of carbohydrate depletion on the structure, stability and activity of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger was purified to homogeneity by hydrophobic interaction and ion-exchange chromatography. Approx. 95% of the carbohydrate moiety was cleaved from the protein by incubation of glucose oxidase with endoglycosidase H and alpha-mannosidase. Cleavage of the carbohydrate moiety effected a 24-30% decrease in the molecular weight and a reduction in the number of isoforms of glucose oxidase. No significant changes were observed in the circular dichroism spectra of the deglycosylated enzyme. Other properties, such as thermal stability, pH and temperature optima of glucose oxidase activity and substrate specificity were not affected. However, removal of the carbohydrate moiety marginally affected the kinetics of glucose oxidation and stability at low pH. From these results it appears that the carbohydrate chain of glucose oxidase does not contribute significantly to the structure, stability and activity of glucose oxidase.

Kinetics and redox-sensitive oligomerisation reveal negative subunit cooperativity in tryparedoxin peroxidase of Trypanosoma brucei brucei.

Abstract Tryparedoxin peroxidases (TXNPx) are peroxiredoxintype enzymes that detoxify hydroperoxides in trypanosomatids. Reduction equivalents are provided by trypanothione [T(SH)2] via tryparedoxin (TXN). The T(SH)2-dependent peroxidase system was reconstituted from TXNPx and TXN of T. brucei brucei (TbTXNPx and TbTXN). TbTXNPx efficiently reduces organic hydroperoxides and is specifically reduced by TbTXN, less efficiently by thioredoxin, but not by glutathione (GSH) or T(SH)2. The kinetic pattern does not comply with a simple rate equation but suggests negative cooperativity of reaction centers. Gel permeation of oxidized TbTXNPx yields peaks corresponding to a decamer and higher aggregates. Electron microscopy shows regular ring structures in the decamer peak. Upon reduction, the rings tend to depolymerise forming openchain oligomers. Co-oxidation of TbTXNPx with TbTXNC43S yields a dead-end intermediate mimicking the catalytic intermediate. Its size complies with a stoichiometry of one TXN per subunit of TXNPx. Electron microscopy of the intermediate displays pentangular structures that are compatible with a model of a decameric TbTXNPx ring with ten bound TbTXN molecules. The redox-dependent changes in shape and aggregation state, the kinetic pattern and molecular models support the view that, upon oxidation of a reaction center, other subunits adopt a conformation that has lower reactivity with the hydroperoxide.

Recombinant tyrosine aminotransferase from Trypanosoma cruzi: structural characterization and site directed mutagenesis of a broad substrate specificity enzyme

The gene encoding tyrosine aminotransferase (TAT, EC 2.6.1.5) from the parasitic protozoan Trypanosoma cruzi was amplified from genomic DNA, cloned into the pET24a expression vector and functionally expressed as a C-terminally His-tagged protein in Escherichia coli BL21(DE3)pLysS. Purified recombinant TAT exhibited identical electrophoretic and enzymatic properties as the authentic enzyme from T. cruzi. Both recombinant and authentic T. cruzi TATs were highly resistant to limited tryptic cleavage and contained no disulfide bonds. Comprehensive analysis of its substrate specificity demonstrated TAT to be a broad substrate aminotransferase, with leucine, methionine as well as tyrosine, phenylalanine, tryptophan and alanine being utilized efficiently as amino donors. Valine, isoleucine and dicarboxylic amino acids served as poor substrates while polar aliphatic amino acids could not be transaminated. TAT also accepted several 2-oxoacids, including 2-oxoisocaproate and 2-oxomethiobutyrate, in addition to pyruvate, oxaloacetate and 2-oxoglutarate. The functionality of the expression system was confirmed by constructing two variants; one (Arg389) being a completely inactive enzyme; the other (Arg283) retaining its full activity, as predicted from the recently solved three-dimensional structure of T. cruzi TAT. Thus, only one of the two strictly conserved arginines which are essential for the enzymatic activity of subfamily Ialpha aspartate and aromatic aminotransferases is critical for T. cruzis TAT activity.

Crystallization and preliminary X-ray diffraction studies of a deglycosylated glucose oxidase from Aspergillus niger.

Deglycosylation was shown to be an important prerequisite step for the crystallization of glucose oxidase from Aspergillus niger. Whereas the glycosylated enzyme could not be crystallized, crystals of the deglycosylated enzyme suitable for X-ray diffraction analysis were reproducibly grown in the presence of 1.6 M-ammonium sulphate and octanetriol at pH 5.3 to 5.6. The crystals belong to the space group P3(1)21 or P3(2)21 with refined lattice constants of a = 66.5 A and c = 214.4 A, indicating a cell content of one monomer per asymmetric unit of the crystal. Crystals diffract to at least 2.5 A resolution. Cleavage of 95% of its carbohydrate moiety affected the kinetics of glucose oxidation, stability at low pH and some electrophoretic properties of glucose oxidase, such as molecular mass and the number of isoelectric forms. However, other properties, such as thermal stability, pH and temperature optima of activity were not affected.

Structures of tryparedoxins revealing interaction with trypanothione.

Abstract Tryparedoxins (TXNs) catalyse the reduction of peroxiredoxin type peroxidases by the bisglutathionyl derivative of spermidine, trypanothione, and are relevant to hydroperoxide detoxification and virulence of trypanosomes. The 3Dstructures of the following tryparedoxins are presented: authentic tryparedoxin1 of Crithidia fasciculata, CfTXN1; the histagged recombinant protein, CfTXN1H6; reduced and oxidised CfTXN2, and an alternative substrate derivative of the mutein CfTXN2H6-Cys44Ser. Cys41 (Cys40 in TXN1) of the active site motif 40-WCPPCR-45 proved to be the only solventexposed redox active residue in CfTXN2. In reduced TXNs, its nucleophilicity is increased by a network of hydrogen bonds. In oxidised TXNs it can be attacked by the thiol of the [1]Nglutathionyl residue of trypanothione, as evidenced by the structure of [1]Nglutathionylspermidinederivatised CfTXN2H6-Cys44Ser. Modelling suggests Arg45 (44), Glu73 (72), the Ile110 (109) cisPro111 (110)bond and Arg129 (128) to be involved in the binding of trypanothione to CfTXN2 (CfTXN1). The model of TXNsubstrate interaction is consistent with functional characteristics of known and newly designed muteins (CfTXN2H6-Arg129Asp and Glu73Arg) and the [1]Nglutathionylspermidine binding in the CfTXN2H6-Cys44Ser structure.

The Structure of a Human Common Cold Virus (Rhinovirus 14) and its Functional Relations to Other Picornaviruses

Picornaviruses are associated with serious diseases in humans and other animals, and they comprise one of the largest families of viral pathogens. For example, the common cold, poliomyelitis, foot-and-mouth disease and hepatitis can be caused by these viruses. They are among the smallest RNA-containing animal viruses (1–3). Their molecular weight is a round 8.5 x 106 and they contain about 30% by weight RNA. Their external diameter is roughly 300 A and they form icosahedral shells. Picornaviridae have been subdivided into four genera on the basis of their buoyant density, pH stability and sedimentation coefficients: enterovirus (e . g . polio, hepatitis A and coxsackie viruses), cardiovirus (e .g . encephalomyocarditis and Mengo viruses), aphthovirus (e.g. foot-and-mouth disease virus) and rhinovirus . They differ also in the number of known serotypes. For instance there are three known serotypes for polioviruses, seven for foot-and- mouth disease viruses (FMDV) and at least 89 for human rhinoviruses (HRV). Accordingly, it has been possible to produce effective vaccines for poliomyelitis and, with greater difficulty , for foot-and-mouth disease, but not for the common cold.

Large-scale purification, enzymic characterization, and crystallization of the lipase from Geotrichum candidum

Lipases from several strains of the imperfect fungus Geotrichum candidum are renowned for their high, though not absolute, specificity for Δ9-unsaturated fatty acids. Starting from a commercially available raw material (GC-4 lipase from Amano), an expeditious two-step isolation method was developed that allowed us to prepare 260 mg of pure enzyme within 2 days. The 61.6-kDa lipase was homogeneous in SDS-PAGE, had a carbohydrate content of 8.8 wt%, and revealed a microheterogeneic pattern between pH 4.4 and 4.8 on isoelectric focusing. Deglycosylation with endoglycosidase H abolished this microheterogeneity, and the lipase focused as a single band at pH 4.63. Over a broad pH range the deglycosylated enzyme had higher specific activities than the native glycosylated form. In hydrolysis as well as in transesterification, a high preference for oleic acid over saturated fatty acids was found for the GC-4 lipase. Crystals were obtained from the native and deglycosylated enzyme as well as from the lipase modified by p-chloromercuribenzoate and iodine. High-quality crystals of native GC-4 lipase grew from media containing polyethylene glycol 20,000. They diffracted to at least 2.5 A, the lattice constants were a = 53.1 A, b = 83.5 A, c = 57.8 A, β = 100° and the space group was monoclinic P 21. A space-filling coefficient of 2.3 A3 was calculated.

Permutation of the active site motif of tryparedoxin 2.

Abstract Tryparedoxins (TXN) are thioredoxinrelated proteins which, as trypanothione:peroxiredoxin oxidoreductases, constitute the trypanothionedependent antioxidant defense and may also serve as substrates for ribonucleotide reductase in trypanosomatids. The active site motif of TXN2, [40]WCPPCR[45], of Crithidia fasciculata was mutated by sitedirected mutagenesis and eight corresponding muteins were expressed in E. coli as terminally Histagged proteins, purified to homogeneity by nickel chelate chromatography, and characterized in terms of specific activity, specificity and, if possible, kinetics. Exchange of Cys41 and Cys44 by serine yielded inactive products confirming their presumed involvement in catalysis. Exchange of Arg45 by aspartate resulted in loss of activity, suggesting an activation of active site cysteines by the positive charge of Arg45. Substitution of Trp40 by phenylalanine or tyrosine resulted in moderate decrease of specific activity, as did exchange of Pro42 by glycine. Kinetic analysis of these three muteins revealed that primarily the reaction with trypanothione is affected by the mutations. Simulation of thioredoxin or glutaredoxin like active sites in TXN2 (P42G and W40T/P43Y, respectively) did not result in thioredoxin or glutaredoxin like activities. These data underscore that TXNs, although belonging to the thioredoxin superfamily, represent a group of enzymes distinct from thioredoxins and glutaredoxins in terms of specificity, and appear attractive as molecular targets for the design of trypanocidal compounds.

Virion orientation in cubic crystals of the human common cold virus HRV14.

A new cubic crystal form (a = 445.1 A) of space group P23 is reported for human rhinovirus R14. There are four particles per unit cell, each situated on a crystallographic 3-fold axis. The orientation of these particles has been determined with a rotation function and their approximate positions have been derived from a Patterson map. The crystals diffract to at least 2.8 A resolution. Limitations to the possible surface features of the virus are set by a comparison of the cubic and orthorhombic crystal forms.