The mitochondrial amidoxime reducing component-from prodrug-activation mechanism to drug-metabolizing enzyme and onward to drug target.

The mitochondrial amidoxime-reducing component (mARC) is one of five known molybdenum enzymes in eukaryotes. mARC belongs to the MOSC domain superfamily, a large group of so far poorly studied molybdoenzymes. mARC was initially discovered as the enzyme activating N-hydroxylated prodrugs of basic amidines but has since been shown to also reduce a variety of other N-oxygenated compounds, for example, toxic nucleobase analogs. Under certain circumstances, mARC might also be involved in reductive nitric oxide synthesis through reduction of nitrite. Recently, mARC enzymes have received a lot of attention due to their apparent involvement in lipid metabolism and, in particular, because many genome-wide association studies have shown a common variant of human mARC1 to have a protective effect against liver disease. The mechanism linking mARC enzymes with lipid metabolism remains unknown. Here, we give a comprehensive overview of what is currently known about mARC enzymes, their substrates, structure, and apparent involvement in human disease.

New Design of an Activity Assay Suitable for High-Throughput Screening of Substrates and Inhibitors of the Mitochondrial Amidoxime Reducing Component (mARC).

The mitochondrial amidoxime-reducing component (mARC) is one of the simplest molybdenum-containing enzymes. mARC is among a few known reducing enzymes playing an important role in drug metabolism in mammals. Here, an assay based on the fluorescence of NADH is reported for the rapid detection of substrates and potential inhibitors of mARC. So far unknown inhibitors might be useful for the development of drugs assigned to nonalcoholic fatty liver disease (NAFLD) and similar diseases. Kinetics of reactions catalyzed by mARC can be recorded with high sensitivity and precision. On a microtiter plate scale, the assay presented could be applied for high-throughput screening of substance libraries and detection of novel mARC substrate candidates. For instance, molnupiravir was also identified as a new substrate by this assay. For better comparison for such substances, the inhibitor or substrate-to-BAO ratio was introduced. After normalization of enzyme activities to the standard benzamidoxime, substrates can reproducibly be classified.

Reduction of Hydrogen Peroxide by Human Mitochondrial Amidoxime Reducing Component Enzymes.

The mitochondrial amidoxime reducing component (mARC) is a human molybdoenzyme known to catalyze the reduction of various N-oxygenated substrates. The physiological function of mARC enzymes, however, remains unknown. In this study, we examine the reduction of hydrogen peroxide (H2O2) by the human mARC1 and mARC2 enzymes. Furthermore, we demonstrate an increased sensitivity toward H2O2 for HEK-293T cells with an MTARC1 knockout, which implies a role of mARC enzymes in the cellular response to oxidative stress. H2O2 is a reactive oxygen species (ROS) formed in all living cells involved in many physiological processes. Furthermore, H2O2 constitutes the first mARC substrate without a nitrogen-oxygen bond, implying that mARC enzymes may have a substrate spectrum going beyond the previously examined N-oxygenated compounds.

WHIRLY1 of Barley and Maize Share a PRAPP Motif Conferring Nucleoid Compaction.

WHIRLY1 in barley was shown to be a major architect of plastid nucleoids. Its accumulation in cells of Escherichia coli coincided with an induction of nucleoid compaction and growth retardation. While WHIRLY1 of maize had similar effects on E. coli cells, WHIRLY1 proteins of Arabidopsis and potato as well as WHIRLY2 proteins had no impact on nucleoid compaction in E. coli. By mutagenesis of HvWHIRLY1 the PRAPP motif at the N-terminus preceding the highly conserved WHIRLY domain was identified to be responsible for the nucleoid compacting activity of HvWHIRLY1 in bacteria. This motif is found in WHIRLY1 proteins of most members of the Poaceae family, but neither in the WHIRLY2 proteins of the family nor in any WHIRLY protein of eudicot species such as Arabidopsis thaliana. This finding indicates that a subset of the monocot WHIRLY1 proteins has acquired a specific function as nucleoid compacters by sequence variation in the N-terminal part preceding the conserved WHIRLY domain and that in different groups of higher plants the compaction of nucleoids is mediated by other proteins.

Crystal structure of human mARC1 reveals its exceptional position among eukaryotic molybdenum enzymes.

Biotransformation enzymes ensure a viable homeostasis by regulating reversible cycles of oxidative and reductive reactions. The metabolism of nitrogen-containing compounds is of high pharmaceutical and toxicological relevance because N-oxygenated metabolites derived from reactions mediated by cytochrome P450 enzymes or flavin-dependent monooxygenases are in some cases highly toxic or mutagenic. The molybdenum-dependent mitochondrial amidoxime-reducing component (mARC) was found to be an extremely efficient counterpart, which is able to reduce the full range of N-oxygenated compounds and thereby mediates detoxification reactions. However, the 3D structure of this enzyme was unknown. Here we present the high-resolution crystal structure of human mARC. We give detailed insight into the coordination of its molybdenum cofactor (Moco), the catalytic mechanism, and its ability to reduce a wide range of N-oxygenated compounds. The identification of two key residues will allow future discrimination between mARC paralogs and ensure correct annotation. Since our structural findings contradict in silico predictions that are currently made by online databases, we propose domain definitions for members of the superfamily of Moco sulfurase C-terminal (MOSC) domain-containing proteins. Furthermore, we present evidence for an evolutionary role of mARC for the emergence of the xanthine oxidase protein superfamily. We anticipate the hereby presented crystal structure to be a starting point for future descriptions of MOSC proteins, which are currently poorly structurally characterized.

Lysozyme's lectin-like characteristics facilitates its immune defense function.

Interactions between human lysozyme (HL) and the lipopolysaccharide (LPS) of Klebsiella pneumoniae O1, a causative agent of lung infection, were identified by surface plasmon resonance. To characterize the molecular mechanism of this interaction, HL binding to synthetic disaccharides and tetrasaccharides representing one and two repeating units, respectively, of the O-chain of this LPS were studied. pH-dependent structural rearrangements of HL after interaction with the disaccharide were observed through nuclear magnetic resonance. The crystal structure of the HL-tetrasaccharide complex revealed carbohydrate chain packing into the A, B, C, and D binding sites of HL, which primarily occurred through residue-specific, direct or water-mediated hydrogen bonds and hydrophobic contacts. Overall, these results support a crucial role of the Glu35/Asp53/Trp63/Asp102 residues in HL binding to the tetrasaccharide. These observations suggest an unknown glycan-guided mechanism that underlies recognition of the bacterial cell wall by lysozyme and may complement the HL immune defense function.

KDEL Receptor 1 Contributes to Cell Surface Association of Protein Disulfide Isomerases.

BACKGROUND/AIMS: Endoplasmic reticulum (ER)-resident proteins with a C-terminal KDEL ERretention sequence are captured in the Golgi apparatus by KDEL receptors (KDELRs). The binding of such proteins to these receptors induces their retrograde transport. Nevertheless, some KDEL proteins, such as Protein Disulfide Isomerases (PDIs), are found at the cell surface. PDIs target disulfide bridges in the extracellular domains of proteins, such as integrins or A Disintegrin And Metalloprotease 17 (ADAM17) leading to changes in the structure and function of these molecules. Integrins become activated and ADAM17 inactivated upon disulfide isomerization. The way that PDIs escape from retrograde transport and reach the plasma membrane remains far from clear. Various mechanisms might exist, depending on whether a local cell surface association or a more global secretion is required. METHODS: To get a more detailed insight in the transport of PDIs to the cell surface, methods such as cell surface biotinylation, flow cytometric analysis, immunoprecipitation, fluorescence microscopy as well as labeling of cells with fluorescence labled recombinant PDIA6 was performed. RESULTS: Here, we show that the C-terminal KDEL ER retention sequence is sufficient to prevent secretion of PDIA6 into the extracellular space but is mandatory for its association with the cell surface. The cell surface trafficking of PDIA1, PDIA3, and PDIA6 is dependent on KDELR1, which travels in a dynamic manner to the cell surface. This transport is assumed to result in PDI cell surface association, which differs from PDI inducible secretion into the extracellular space. Distinct PDIs differ in their trafficking properties. Endogenous KDELR1, detectable at the cell surface, might be involved not only in the transport of cell-surface-associated PDIs, but also in their retrieval and internalization from the extracellular space. CONCLUSION: Beside their ER retention motive PDIs travel to the cell surface. Here they target different proteins to render their function. To escape the ER PDIs travel via various pathways. One of them depends on the KDELR1, which can transport its target to the cell surface, where it is to be expected to release its cargo in close vicinity to its target molecules. Hence, the KDEL sequence is needed for cell surface association of PDIs, such as PDIA6.

Structure of NDP-forming Acetyl-CoA synthetase ACD1 reveals a large rearrangement for phosphoryl transfer.

The NDP-forming acyl-CoA synthetases (ACDs) catalyze the conversion of various CoA thioesters to the corresponding acids, conserving their chemical energy in form of ATP. The ACDs are the major energy-conserving enzymes in sugar and peptide fermentation of hyperthermophilic archaea. They are considered to be primordial enzymes of ATP synthesis in the early evolution of life. We present the first crystal structures, to our knowledge, of an ACD from the hyperthermophilic archaeon Candidatus Korachaeum cryptofilum. These structures reveal a unique arrangement of the ACD subunits alpha and beta within an α2ß2-heterotetrameric complex. This arrangement significantly differs from other members of the superfamily. To transmit an activated phosphoryl moiety from the Ac-CoA binding site (within the alpha subunit) to the NDP-binding site (within the beta subunit), a distance of 51 Å has to be bridged. This transmission requires a larger rearrangement within the protein complex involving a 21-aa-long phosphohistidine-containing segment of the alpha subunit. Spatial restraints of the interaction of this segment with the beta subunit explain the necessity for a second highly conserved His residue within the beta subunit. The data support the proposed four-step reaction mechanism of ACDs, coupling acyl-CoA thioesters with ATP synthesis. Furthermore, the determined crystal structure of the complex with bound Ac-CoA allows first insight, to our knowledge, into the determinants for acyl-CoA substrate specificity. The composition and size of loops protruding into the binding pocket of acyl-CoA are determined by the individual arrangement of the characteristic subdomains.

The Involvement of the Mitochondrial Amidoxime Reducing Component (mARC) in the Reductive Metabolism of Hydroxamic Acids.

The mitochondrial amidoxime reducing component is a recently discovered molybdenum enzyme in mammals which, in concert with the electron transport proteins cytochrome b5 and NADH cytochrome b5 reductase, catalyzes the reduction of N-oxygenated structures. This three component enzyme system plays a major role in N-reductive drug metabolism. Belonging to the group of N-hydroxylated structures, hydroxamic acids are also potential substrates of the mARC-system. Hydroxamic acids show a variety of pharmacological activities and are therefore often found in drug candidates. They can also exhibit toxic properties as is the case for many aryl hydroxamic acids formed during the metabolism of arylamides. Biotransformation assays using recombinant human proteins, subcellular porcine tissue fractions as well as human cell culture were performed. Here the mARC-dependent reduction of the model compound benzhydroxamic acid is reported in addition to the reduction of three drugs. In comparison with other known substrates of the molybdenum depending enzyme system (e.g., amidoxime prodrugs) the conversion rates measured here are slower, thereby reflecting the mediocre metabolic stability and oral bioavailability of distinct hydroxamic acids. Moreover, the toxic N-hydroxylated metabolite of the analgesic phenacetin, N-hydroxyphenacetin, is not reduced by the mARC-system under the chosen conditions. This confirms the high toxicity of this component, as it needs to be detoxified by other pathways. This work highlights the need to monitor the N-reductive metabolism of new drug candidates by the mARC-system when evaluating the metabolic stability of hydroxamic acid-containing structures or the potential risks of toxic metabolites.

Beta-aminoketones as prodrugs for selective irreversible inhibitors of type-1 methionine aminopeptidases.

We identified and characterized ß-aminoketones as prodrugs for irreversible MetAP inhibitors that are selective for the MetAP-1 subtype. ß-Aminoketones with certain structural features form α,ß-unsaturated ketones under physiological conditions, which bind covalently and selectively to cysteines in the S1 pocket of MetAP-1. The binding mode was confirmed by X-ray crystallography and assays with the MetAPs from Escherichia coli, Staphylococcus aureus and both human isoforms. The initially identified tetralone derivatives showed complete selectivity for E. coli MetAP versus human MetAP-1 and MetAP-2. Rational design of indanone analogs yielded compounds with selectivity for the human type-1 versus the human type-2 MetAP.

mARC1 in MASLD: Modulation of lipid accumulation in human hepatocytes and adipocytes.

BACKGROUND: Mutations in the gene MTARC1 (mitochondrial amidoxime-reducing component 1) protect carriers from metabolic dysfunction-associated steatohepatitis (MASH) and cirrhosis. MTARC1 encodes the mARC1 enzyme, which is localized to the mitochondria and has no known MASH-relevant molecular function. Our studies aimed to expand on the published human genetic mARC1 data and to observe the molecular effects of mARC1 modulation in preclinical MASH models. METHODS AND RESULTS: We identified a novel human structural variant deletion in MTARC1, which is associated with various biomarkers of liver health, including alanine aminotransferase levels. Phenome-wide Mendelian Randomization analyses additionally identified novel putatively causal associations between MTARC1 expression, and esophageal varices and cardiorespiratory traits. We observed that protective MTARC1 variants decreased protein accumulation in in vitro overexpression systems and used genetic tools to study mARC1 depletion in relevant human and mouse systems. Hepatocyte mARC1 knockdown in murine MASH models reduced body weight, liver steatosis, oxidative stress, cell death, and fibrogenesis markers. mARC1 siRNA treatment and overexpression modulated lipid accumulation and cell death consistently in primary human hepatocytes, hepatocyte cell lines, and primary human adipocytes. mARC1 depletion affected the accumulation of distinct lipid species and the expression of inflammatory and mitochondrial pathway genes/proteins in both in vitro and in vivo models. CONCLUSIONS: Depleting hepatocyte mARC1 improved metabolic dysfunction-associated steatotic liver disease-related outcomes. Given the functional role of mARC1 in human adipocyte lipid accumulation, systemic targeting of mARC1 should be considered when designing mARC1 therapies. Our data point to plasma lipid biomarkers predictive of mARC1 abundance, such as Ceramide 22:1. We propose future areas of study to describe the precise molecular function of mARC1, including lipid trafficking and subcellular location within or around the mitochondria and endoplasmic reticulum.

Mass spectrometric analysis of hepatitis C viral envelope protein E2 reveals extended microheterogeneity of mucin-type O-linked glycosylation.

The infectious liver disease hepatitis C is caused by the small, enveloped, positive single-strand RNA hepatitis C virus (HCV). The HCV genome encodes for a single polyprotein precursor of ∼3010 amino acid residues. Host and cellular proteases co- and posttranslational process the precursor creating six nonstructural (NS) proteins and four structural components. Properly folded forms of the envelope proteins E1 and E2 form the associated E1-E2 complex. This complex represents a significant antigenic component at the viral surface that can interact with several target cell receptors. Extent and type of glycosylation is an important factor for virulence and escape from the immune system. Detailed characterization of the glycosylated sites is helpful for the understanding of different phenotypes as well as for the development of E1/E2-related treatments of HCV infection. Here, we have investigated in detail the O-linked glycosylation of the HCV envelope protein E2 expressed in and isolated from human embryonic kidney (HEK 293) cells. Using nano-liquid chromatography and tandem mass spectrometry approaches, we clearly have identified six residues for O-linked glycosylation within isolated glycopeptides (Ser393, Thr396, Ser401, Ser404, Thr473 and Thr518), carrying mainly Core 1 and Core 2 mucin-type structures. Based on our data, Thr385 is probably glycosylated as well. In addition, we could show that Ser479 within the hyper variable region (HVR) I is not O-glycosylated. For most of these sites, different degrees of microheterogeneity could be verified. Concerning HCV E2, this is the first case of experimentally proven O-linked glycosylation in detail via mass spectrometry.

EcoTILLING in Beta vulgaris reveals polymorphisms in the FLC-like gene BvFL1 that are associated with annuality and winter hardiness.

BACKGROUND: Sugar beet (Beta vulgaris ssp. vulgaris L.) is an important crop for sugar and biomass production in temperate climate regions. Currently sugar beets are sown in spring and harvested in autumn. Autumn-sown sugar beets that are grown for a full year have been regarded as a cropping system to increase the productivity of sugar beet cultivation. However, for the development of these "winter beets" sufficient winter hardiness and a system for bolting control is needed. Both require a thorough understanding of the underlying genetics and its natural variation. RESULTS: We screened a diversity panel of 268 B. vulgaris accessions for three flowering time genes via EcoTILLING. This panel had been tested in the field for bolting behaviour and winter hardiness. EcoTILLING identified 20 silent SNPs and one non-synonymous SNP within the genes BTC1, BvFL1 and BvFT1, resulting in 55 haplotypes. Further, we detected associations of nucleotide polymorphisms in BvFL1 with bolting before winter as well as winter hardiness. CONCLUSIONS: These data provide the first genetic indication for the function of the FLC homolog BvFL1 in beet. Further, it demonstrates for the first time that EcoTILLING is a powerful method for exploring genetic diversity and allele mining in B. vulgaris.

The structure of substrate-free 1,5-anhydro-D-fructose reductase from Sinorhizobium meliloti 1021 reveals an open enzyme conformation.

1,5-Anhydro-D-fructose (1,5-AF) is an interesting building block for enantioselective and stereoselective organic synthesis. Enzymes acting on this compound are potential targets for structure-based protein/enzyme design to extend the repertoire of catalytic modifications of this and related building blocks. Recombinant 1,5-anhydro-D-fructose reductase (AFR) from Sinorhizobium meliloti 1021 was produced in Escherichia coli, purified using a fused 6×His affinity tag and crystallized in complex with the cofactor NADP(H) using the hanging-drop technique. Its structure was determined to 1.93 Å resolution using molecular replacement. The structure displays an empty substrate-binding site and can be interpreted as an open conformation reflecting the enzyme state shortly after the release of product, presumably with bound oxidized cofactor NADP⁺. Docking simulations indicated that amino-acid residues Lys94, His151, Trp162, Arg163, Asp176 and His180 are involved in substrate binding, catalysis or product release. The side chain of Lys94 seems to have the ability to function as a molecular switch. The crystal structure helps to characterize the interface relevant for dimer formation as observed in solution. The crystal structure is compared with the structure of the homologue from S. morelense, which was solved in a closed conformation and for which dimer formation in solution could not be verified but seems to be likely based on the presented studies of S. meliloti AFR.

Structural basis for the oxidation of protein-bound sulfur by the sulfur cycle molybdohemo-enzyme sulfane dehydrogenase SoxCD.

The sulfur cycle enzyme sulfane dehydrogenase SoxCD is an essential component of the sulfur oxidation (Sox) enzyme system of Paracoccus pantotrophus. SoxCD catalyzes a six-electron oxidation reaction within the Sox cycle. SoxCD is an α(2)ß(2) heterotetrameric complex of the molybdenum cofactor-containing SoxC protein and the diheme c-type cytochrome SoxD with the heme domains D(1) and D(2). SoxCD(1) misses the heme-2 domain D(2) and is catalytically as active as SoxCD. The crystal structure of SoxCD(1) was solved at 1.33 Å. The substrate of SoxCD is the outer (sulfane) sulfur of Cys-110-persulfide located at the C-terminal peptide swinging arm of SoxY of the SoxYZ carrier complex. The SoxCD(1) substrate funnel toward the molybdopterin is narrow and partially shielded by side-chain residues of SoxD(1). For access of the sulfane-sulfur of SoxY-Cys-110 persulfide we propose that (i) the blockage by SoxD-Arg-98 is opened via interaction with the C terminus of SoxY and (ii) the C-terminal peptide VTIGGCGG of SoxY provides interactions with the entrance path such that the cysteine-bound persulfide is optimally positioned near the molybdenum atom. The subsequent oxidation reactions of the sulfane-sulfur are initiated by the nucleophilic attack of the persulfide anion on the molybdenum atom that is, in turn, reduced. The close proximity of heme-1 to the molybdopterin allows easy acceptance of the electrons. Because SoxYZ, SoxXA, and SoxB are already structurally characterized, with SoxCD(1) the structures of all key enzymes of the Sox cycle are known with atomic resolution.

Identification of New Antibodies Targeting Malignant Plasma Cells for Immunotherapy by Next-Generation Sequencing-Assisted Phage Display.

To identify new antibodies for the treatment of plasma cell disorders including multiple myeloma (MM), a single-chain Fragment variable (scFv) antibody library was generated by immunizing mice with patient-derived malignant plasma cells. To enrich antibodies binding myeloma antigens, phage display with cellular panning was performed. After depleting the immune library with leukocytes of healthy donors, selection of antibodies was done with L-363 plasma cell line in two consecutive panning rounds. Monitoring the antibodies' enrichment throughout the panning by next-generation sequencing (NGS) identified several promising candidates. Initially, 41 unique scFv antibodies evolving from different B cell clones were selected. Nine of these antibodies strongly binding to myeloma cells and weakly binding to peripheral blood mononuclear cells (PBMC) were characterized. Using stably transfected Chinese hamster ovary cells expressing individual myeloma-associated antigens revealed that two antibodies bind CD38 and intercellular adhesion molecule-1 (ICAM-1), respectively, and 7 antibodies target yet unknown antigens. To evaluate the therapeutic potential of our new antibodies, in a first proof-of-concept study the CD38 binding scFv phage antibody was converted into a chimeric IgG1. Further analyses revealed that #5-CD38-IgG1 shared an overlapping epitope with daratumumab and isatuximab and had potent anti-myeloma activity comparable to the two clinically approved CD38 antibodies. These results indicate that by phage display and deep sequencing, new antibodies with therapeutic potential for MM immunotherapy can be identified.

Structural insight into substrate differentiation of the sugar-metabolizing enzyme galactitol dehydrogenase from Rhodobacter sphaeroides D.

Galactitol 2-dehydrogenase (GatDH) belongs to the protein superfamily of short-chain dehydrogenases. As an enzyme capable of the stereo- and regioselective modification of carbohydrates, it exhibits a high potential for application in biotechnology as a biocatalyst. We have determined the crystal structure of the binary form of GatDH in complex with its cofactor NAD(H) and of the ternary form in complex with NAD(H) and three different substrates. The active form of GatDH constitutes a homo-tetramer with two magnesium-ion binding sites each formed by two opposing C termini. The catalytic tetrad is formed by Asn(116), Ser(144), Tyr(159), and Lys(163). GatDH structurally aligns well with related members of the short-chain dehydrogenase family. The substrate binding pocket can be divided into two parts of different size and polarity. In the smaller part, the side chains of amino acids Ser(144), Ser(146), and Asn(151) are important determinants for the binding specificity and the orientation of (pro-) chiral compounds. The larger part of the pocket is elongated and flanked by polar and non-polar residues, enabling a rather broad substrate spectrum. The presented structures provide valuable information for a rational design of this enzyme to improve its stability against pH, temperature, or solvent concentration and to optimize product yield in bioreactors.

Structural and catalytic characterization of Blastochloris viridis and Pseudomonas aeruginosa homospermidine synthases supports the essential role of cation-π interaction.

Polyamines influence medically relevant processes in the opportunistic pathogen Pseudomonas aeruginosa, including virulence, biofilm formation and susceptibility to antibiotics. Although homospermidine synthase (HSS) is part of the polyamine metabolism in various strains of P. aeruginosa, neither its role nor its structure has been examined so far. The reaction mechanism of the nicotinamide adenine dinucleotide (NAD+)-dependent bacterial HSS has previously been characterized based on crystal structures of Blastochloris viridis HSS (BvHSS). This study presents the crystal structure of P. aeruginosa HSS (PaHSS) in complex with its substrate putrescine. A high structural similarity between PaHSS and BvHSS with conservation of the catalytically relevant residues is demonstrated, qualifying BvHSS as a model for mechanistic studies of PaHSS. Following this strategy, crystal structures of single-residue variants of BvHSS are presented together with activity assays of PaHSS, BvHSS and BvHSS variants. For efficient homospermidine production, acidic residues are required at the entrance to the binding pocket (`ionic slide') and near the active site (`inner amino site') to attract and bind the substrate putrescine via salt bridges. The tryptophan residue at the active site stabilizes cationic reaction components by cation-π interaction, as inferred from the interaction geometry between putrescine and the indole ring plane. Exchange of this tryptophan for other amino acids suggests a distinct catalytic requirement for an aromatic interaction partner with a highly negative electrostatic potential. These findings substantiate the structural and mechanistic knowledge on bacterial HSS, a potential target for antibiotic design.

New insight into the dynamic properties and the active site architecture of H-Ras p21 revealed by X-ray crystallography at very high resolution.

BACKGROUND: In kinetic crystallography, the usually static method of X-ray diffraction is expanded to allow time-resolved analysis of conformational rearrangements in protein structures. To achieve this, reactions have to be triggered within the protein crystals of interest, and optical spectroscopy can be used to monitor the reaction state. For this approach, a modified form of H-Ras p21 was designed which allows reaction initiation and fluorescence readout of the initiated GTPase reaction within the crystalline state. Rearrangements within the crystallized protein due to the progressing reaction and associated heterogeneity in the protein conformations have to be considered in the subsequent refinement processes. RESULTS: X-ray diffraction experiments on H-Ras p21 in different states along the reaction pathway provide detailed information about the kinetics and mechanism of the GTPase reaction. In addition, a very high data quality of up to 1.0 Å resolution allowed distinguishing two discrete subconformations of H-Ras p21, expanding the knowledge about the intrinsic flexibility of Ras-like proteins, which is important for their function. In a complex of H-Ras•GppNHp (guanosine-5'-(ß,γ-imido)-triphosphate), a second Mg2+ ion was found to be coordinated to the γ-phosphate group of GppNHp, which positions the hydrolytically active water molecule very close to the attacked γ-phosphorous atom. CONCLUSION: For the structural analysis of very high-resolution data we have used a new 'two-chain-isotropic-refinement' strategy. This refinement provides an alternative and easy to interpret strategy to reflect the conformational variability within crystal structures of biological macromolecules. The presented fluorescent form of H-Ras p21 will be advantageous for fluorescence studies on H-Ras p21 in which the use of fluorescent nucleotides is not feasible.

Preliminary X-ray crystallographic analysis of the D-xylulose 5-phosphate phosphoketolase from Lactococcus lactis.

Phosphoketolases are thiamine diphosphate-dependent enzymes which play a central role in the pentose-phosphate pathway of heterofermentative lactic acid bacteria. They belong to the family of aldehyde-lyases and in the presence of phosphate ion cleave the carbon-carbon bond of the specific substrate D-xylulose 5-phosphate (or D-fructose 6-phosphate) to give acetyl phosphate and D-glyceraldehyde 3-phosphate (or D-erythrose 4-phosphate). Structural information about phosphoketolases is particularly important in order to fully understand their mechanism as well as the steric course of phosphoketolase-catalyzed reactions. Here, the purification, preliminary crystallization and crystallographic characterization of D-xylulose 5-phosphate phosphoketolase from Lactococcus lactis are reported. The presence of thiamine diphosphate during purification was essential for the enzymatic activity of the purified protein. The crystals belonged to the monoclinic space group P2(1). Diffraction data were obtained to a resolution of 2.2 A.