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A study of in vitro refolding and isotope effects on protein structure, activity and stability shows that different folding dynamics can lead to important changes in protein properties., The biological function of a protein is intimately related to its structure and dynamics, which in turn are determined by the way in which it has been folded. In vitro refolding is commonly used for the recovery of recombinant proteins that are expressed in the form of inclusion bodies and is of central interest in terms of the folding pathways that occur in vivo. Here, biophysical data are reported for in vitro-refolded hydrogenated hen egg-white lysozyme, in combination with atomic resolution X-ray diffraction analyses, which allowed detailed comparisons with native hydrogenated and refolded perdeuterated lysozyme. Distinct folding modes are observed for the hydrogenated and perdeuterated refolded variants, which are determined by conformational changes to the backbone structure of the Lys97–Gly104 flexible loop. Surprisingly, the structure of the refolded perdeuterated protein is closer to that of native lysozyme than that of the refolded hydrogenated protein. These structural differences suggest that the observed decreases in thermal stability and enzymatic activity in the refolded perdeuterated and hydrogenated proteins are consequences of the macromolecular deuteration effect and of distinct folding dynamics, respectively. These results are discussed in the context of both in vitro and in vivo folding, as well as of lysozyme amyloidogenesis.

Perdeuteration and in vitro refolding of hen egg-white lysozyme impact protein thermal stability and activity. Deuteration appears to primarily affect enzymatic function through changes in protein dynamics, while refolding contributes to a small decrease in protein thermal stability., This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression of insoluble protein in Escherichia coli followed by in-column chemical refolding. This allowed detailed comparisons with perdeuterated lysozyme produced in the yeast Pichia pastoris, as well as with unlabelled lysozyme. Both perdeuterated variants exhibit reduced thermal stability and enzymatic activity in comparison with hydrogenated lysozyme. The thermal stability of refolded perdeuterated lysozyme is 4.9°C lower than that of the perdeuterated variant expressed and secreted in yeast and 6.8°C lower than that of the hydrogenated Gallus gallus protein. However, both perdeuterated variants exhibit a comparable activity. Atomic resolution X-ray crystallographic analyses show that the differences in thermal stability and enzymatic function are correlated with refolding and deuteration effects. The hydrogen/deuterium isotope effect causes a decrease in the stability and activity of the perdeuterated analogues; this is believed to occur through a combination of changes to hydrophobicity and protein dynamics. The lower level of thermal stability of the refolded perdeuterated lysozyme is caused by the unrestrained Asn103 peptide-plane flip during the unfolded state, leading to a significant increase in disorder of the Lys97–Gly104 region following subsequent refolding. An ancillary outcome of this study has been the development of an efficient and financially viable protocol that allows stable and active perdeuterated lysozyme to be more easily available for scientific applications.

Nature Communications 11(1), 1026 (2020). doi:10.1038/s41467-020-14833-9, Structural and functional studies were conducted of the glucuronoyl esterase (GE) from Cerrena unicolor (CuGE), an enzyme catalyzing cleavage of lignin-carbohydrate ester bonds. CuGE is an α/β-hydrolase belonging to carbohydrate esterase family 15 (CE15). The enzyme is modular, comprised of a catalytic and a carbohydrate-binding domain. SAXS data show CuGE as an elongated rigid molecule where the two domains are connected by a rigid linker. Detailed structural information of the catalytic domain in its apo- and inactivated form and complexes with aldouronic acids reveal well-defined binding of the 4-O-methyl-a-D-glucuronoyl moiety, not influenced by the nature of the attached xylo-oligosaccharide. Structural and sequence comparisons within CE15 enzymes reveal two distinct structural subgroups. CuGE belongs to the group of fungal CE15-B enzymes with an open and flat substrate-binding site. The interactions between CuGE and its natural substrates are explained and rationalized by the structural results, microscale thermophoresis and isothermal calorimetry., Published by Nature Publishing Group UK, [London]

Significance Ink, invented in ancient Egypt circa 5,000 y ago, is the established and time-honored medium wherewith humankind commits words to writing. A comprehensive synchrotron-based microanalysis of a considerable corpus of ancient Egyptian papyri from the Roman period, inscribed with red and black inks, reveal a hitherto undetected complex composition of inks. Highlighted by the presence of iron, the red color can be attributed to the use of ocher. Unexpectedly, lead is regularly present in both the red and black inks and is associated to phosphate, sulfate, chloride, and carboxylate ions. The analysis shows that lead was probably used as a drier rather than as a pigment, similar to its usage in 15th century Europe during the development of oil paintings., A hitherto unknown composition is highlighted in the red and black inks preserved on ancient Egyptian papyri from the Roman period (circa 100 to 200 CE). Synchrotron-based macro–X-ray fluorescence (XRF) mapping brings to light the presence of iron (Fe) and lead (Pb) compounds in the majority of the red inks inscribed on 12 papyrus fragments from the Tebtunis temple library. The iron-based compounds in the inks can be assigned to ocher, notably due to the colocalization of Fe with aluminum, and the detection of hematite (Fe2O3) by micro–X-ray diffraction. Using the same techniques together with micro-Fourier transform infrared spectroscopy, Pb is shown to be associated with fatty acid phosphate, sulfate, chloride, and carboxylate ions. Moreover, micro-XRF maps reveal a peculiar distribution and colocalization of Pb, phosphorus (P), and sulfur (S), which are present at the micrometric scale resembling diffused “coffee rings” surrounding the ocher particles imbedded in the red letters, and at the submicrometric scale concentrated in the papyrus cell walls. A similar Pb, P, and S composition was found in three black inks, suggesting that the same lead components were employed in the manufacture of carbon-based inks. Bearing in mind that pigments such as red lead (Pb3O4) and lead white (hydrocerussite [Pb3(CO3)2(OH)2] and/or cerussite [PbCO3]) were not detected, the results presented here suggest that the lead compound in the ink was used as a drier rather than as a pigment. Accordingly, the study calls for a reassessment of the composition of lead-based components in ancient Mediterranean pigments.

CAZypedia was initiated in 2007 to create a comprehensive, living encyclopedia of the carbohydrate-active enzymes (CAZymes) and associated carbohydrate-binding modules involved in the synthesis, modification, and degradation of complex carbohydrates. CAZypedia is closely connected with the actively-curated CAZy database, which provides a sequence-based foundation for the biochemical, mechanistic, and structural characterization of these diverse proteins. Now celebrating its 10th anniversary online, CAZypedia is a successful example of dynamic, community-driven, and expert-based biocuration. CAZypedia is an open-access resource available at URL http://www.cazypedia.org.

A new algorithm automatically determines the structures of large macromolecules of unknown fold from low-resolution single-wavelength anomalous X-ray data and a partial model that failed with other methods., Determining macromolecular structures from X-ray data with resolution worse than 3 Å remains a challenge. Even if a related starting model is available, its incompleteness or its bias together with a low observation-to-parameter ratio can render the process unsuccessful or very time-consuming. Yet, many biologically important macromolecules, especially large macromolecular assemblies, membrane proteins and receptors, tend to provide crystals that diffract to low resolution. A new algorithm to tackle this problem is presented that uses a multivariate function to simultaneously exploit information from both an initial partial model and low-resolution single-wavelength anomalous diffraction data. The new approach has been used for six challenging structure determinations, including the crystal structures of membrane proteins and macromolecular complexes that have evaded experts using other methods, and large structures from a 3.0 Å resolution F1-ATPase data set and a 4.5 Å resolution SecYEG–SecA complex data set. All of the models were automatically built by the method to R free values of between 28.9 and 39.9% and were free from the initial model bias.

For the first time it is shown that carbon black inks on ancient Egyptian papyri from different time periods and geographical regions contain copper. The inks have been investigated using synchrotron-based micro X-ray fluorescence (XRF) and micro X-ray absorption near-edge structure spectroscopy (XANES) at the European Synchrotron Radiation Facility (ESRF). The composition of the copper-containing carbon inks showed no significant differences that could be related to time periods or the geographical locations. This renders it probable that the same technology for ink production was used throughout Egypt for a period spanning at least 300 years. It is argued that the black pigment material (soot) for these inks was obtained as by-products of technical metallurgy. The copper (Cu) can be correlated with the following three main components: cuprite (Cu2O), azurite (Cu3[CO3]2[OH]2) and malachite (Cu2CO3[OH]2).

The metallonuclease colicin E7 is a member of the HNH family of endonucleases. It serves as a bacterial toxin in Escherichia coli, protecting the host cell from other related bacteria and bacteriophages by degradation of their chromosomal DNA under environmental stress. Its cell-killing activity is attributed to the nonspecific nuclease domain (NColE7), which possesses the catalytic ββα-type metal ion-binding HNH motif at its C-terminus. Mutations affecting the positively charged amino acids at the N-terminus of NColE7 (444–­576) surprisingly showed no or significantly reduced endonuclease activity [Czene et al. (2013 ▶), J. Biol. Inorg. Chem. 18, 309–321]. The necessity of the N-­terminal amino acids for the function of the C-terminal catalytic centre poses the possibility of allosteric activation within the enzyme. Precise knowledge of the intramolecular interactions of these residues that affect the catalytic activity could turn NColE7 into a novel platform for artificial nuclease design. In this study, the N-terminal deletion mutant ΔN4-NColE7-C* of the nuclease domain of colicin E7 selected by E. coli was overexpressed and crystallized at room temperature by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.6 A resolution and could be indexed and averaged in the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 55.4, c = 73.1 A. Structure determination by molecular replacement is in progress.

Background: PfEMP1 proteins cause Plasmodium falciparum-infected erythrocytes to bind human tissues during malaria. Results: The IT4VAR13 ectodomain is rigid, elongated, and monomeric, presenting a binding site for its ligand, ICAM-1. Conclusion: The IT4VAR13 ectodomain is unlike that of VAR2CSA, a PfEMP1 that adopts a compact structure with multiple domains contributing to ligand binding. Significance: PfEMP1 proteins have evolved diverse architectures to facilitate ligand recognition., The adhesion of Plasmodium falciparum-infected erythrocytes to human tissues or endothelium is central to the pathology caused by the parasite during malaria. It contributes to the avoidance of parasite clearance by the spleen and to the specific pathologies of cerebral and placental malaria. The PfEMP1 family of adhesive proteins is responsible for this sequestration by mediating interactions with diverse human ligands. In addition, as the primary targets of acquired, protective immunity, the PfEMP1s are potential vaccine candidates. PfEMP1s contain large extracellular ectodomains made from CIDR (cysteine-rich interdomain regions) and DBL (Duffy-binding-like) domains and show extensive variation in sequence, size, and domain organization. Here we use biophysical methods to characterize the entire ∼300-kDa ectodomain from IT4VAR13, a protein that interacts with the host receptor, intercellular adhesion molecule-1 (ICAM-1). We show through small angle x-ray scattering that IT4VAR13 is rigid, elongated, and monomeric. We also show that it interacts with ICAM-1 through the DBLβ domain alone, forming a 1:1 complex. These studies provide a first low resolution structural view of a PfEMP1 ectodomain in complex with its ligand. They show that it combines a modular domain arrangement consisting of individual ligand binding domains, with a defined higher order architecture that exposes the ICAM-1 binding surface to allow adhesion.

X-ray diffraction data of high quality measured to high resolution on crystals of the two pentitol epimers ribitol (centric) and xylitol (acentric) at 101, 141, and 181 K and data on the two compounds previously recorded at 122 K have formed the basis for multipole refinements with the VALRAY system. Our analysis showed that it is possible to obtain a reliable crystal electron density for an acentric compound (xylitol) from X-ray diffraction data and that the thermal motion can be deconvoluted from the static density in this temperature range. The Bader-type topological analysis of the static electron densities revealed virtually identical intramolecular interactions as well as very similar hydrogen bond interactions of ribitol and xylitol; the only minor differences are found in the weaker intermolecular interactions. The high-level periodic DFT calculations are in accordance with the thermodynamic measurements that show that the two pentitols have identical sublimation energies. A rigid body normal coordinate analysis was performed on the atomic displacement parameters obtained at the four different temperatures. The translational and librational mean square deviations derived through this analysis were used in a quantum statistical approach to derive frequencies of the corresponding harmonic oscillators. The analysis showed a consistent vibrational model for all temperatures. The frequencies were subsequently used to calculate crystal entropies assuming an Einstein-type behavior. These calculations show that the crystal entropy of ribitol is 8 J K(-1) mol(-1) higher than the crystal entropy of xylitol, confirming that it is a difference in the entropy of the two compounds that causes the difference in their free energy. Our results presented in this Article show the potential to use X-ray diffraction data to obtain physicochemical properties of crystals.

Laccases of different biological origins have been widely investigated and these studies have elucidated fundamentals of the generic catalytic mechanism. However, other features such as surface properties and residues located away from the catalytic centres may also have impact on enzyme function. Here we present the crystal structure of laccase from Myceliophthora thermophila (MtL) to a resolution of 1.62 Å together with a thorough structural comparison with other members of the CAZy family AA1_3 that comprises fungal laccases from ascomycetes. The recombinant protein produced in A. oryzae has a molecular mass of 75 kDa, a pI of 4.2 and carries 13.5 kDa N-linked glycans. In the crystal, MtL forms a dimer with the phenolic substrate binding pocket blocked, suggesting that the active form of the enzyme is monomeric. Overall, the MtL structure conforms with the canonical fold of fungal laccases as well as the features specific for the asco-laccases. However, the structural comparisons also reveal significant variations within this taxonomic subgroup. Notable differences in the T1-Cu active site topology and polar motifs imply molecular evolution to serve different functional roles. Very few surface residues are conserved and it is noticeable that they encompass residues that interact with the N-glycans and/or are located at domain interfaces. The N-glycosylation sites are surprisingly conserved among asco-laccases and in most cases the glycan displays extensive interactions with the protein. In particular, the glycans at Asn88 and Asn210 appear to have evolved as an integral part of the asco-laccase structure. An uneven distribution of the carbohydrates around the enzyme give unique properties to a distinct part of the surface of the asco-laccases which may have implication for laccase function–in particular towards large substrates.

We present here the first experimental evidence for bound substrate in the active site of a rhamnogalacturonan lyase belonging to family 4 of polysaccharide lyases, Aspergillus aculeatus rhamnogalacturonan lyase (RGL4). RGL4 is involved in the degradation of rhamnogalacturonan-I, an important pectic plant cell wall polysaccharide. Based on the previously determined wild-type structure, enzyme variants RGL4_H210A and RGL4_K150A have been produced and characterized both kinetically and structurally, showing that His210 and Lys150 are key active-site residues. Crystals of the RGL4_K150A variant soaked with a rhamnogalacturonan digest gave a clear picture of substrate bound in the − 3/+ 3 subsites. The crystallographic and kinetic studies on RGL4, and structural and sequence comparison to other enzymes in the same and other PL families, enable us to propose a detailed reaction mechanism for the β-elimination on [-,2)-α- l -rhamno-(1,4)-α- d -galacturonic acid-(1,-]. The mechanism differs significantly from the one established for pectate lyases, in which most often calcium ions are engaged in catalysis.

The short hydrogen bonds in rhamnogalacturonan acetylesterase have been investigated by structure determination of an active-site mutant, 1H NMR spectra and computational methods. Comparisons are made to database statistics. A very short carboxylic acid carboxylate hydrogen bond, buried in the protein, could explain the low-field (18 p.p.m.) 1H NMR signal., An extremely low-field signal (at approximately 18 p.p.m.) in the 1H NMR spectrum of rhamnogalacturonan acetylesterase (RGAE) shows the presence of a short strong hydrogen bond in the structure. This signal was also present in the mutant RGAE D192N, in which Asp192, which is part of the catalytic triad, has been replaced with Asn. A careful analysis of wild-type RGAE and RGAE D192N was conducted with the purpose of identifying possible candidates for the short hydrogen bond with the 18 p.p.m. deshielded proton. Theor­etical calculations of chemical shift values were used in the interpretation of the experimental 1H NMR spectra. The crystal structure of RGAE D192N was determined to 1.33 Å resolution and refined to an R value of 11.6% for all data. The structure is virtually identical to the high-resolution (1.12 Å) structure of the wild-type enzyme except for the interactions involving the mutation and a disordered loop. Searches of the Cambridge Structural Database were conducted to obtain information on the donor–acceptor distances of different types of hydrogen bonds. The short hydrogen-bond inter­actions found in RGAE have equivalents in small-molecule structures. An examination of the short hydrogen bonds in RGAE, the calculated pK a values and solvent-accessibilities identified a buried carboxylic acid carboxylate hydrogen bond between Asp75 and Asp87 as the likely origin of the 18 p.p.m. signal. Similar hydrogen-bond interactions between two Asp or Glu carboxy groups were found in 16% of a homology-reduced set of high-quality structures extracted from the PDB. The shortest hydrogen bonds in RGAE are all located close to the active site and short interactions between Ser and Thr side-chain OH groups and backbone carbonyl O atoms seem to play an important role in the stability of the protein structure. These results illustrate the significance of short strong hydrogen bonds in proteins.

Anisotropic displacement parameters (ADPs) are compared for H atoms estimated using three recently described procedures, both among themselves and with neutron diffraction results. The results convincingly demonstrate that all methods are capable of giving excellent results for several benchmark systems and identify systematic discrepancies for several atom types. A revised and extended library of internal H-atom mean-square displacements is presented for use with Madsen's SHADE web server [J. Appl. Cryst. (2006), 39, 757–758; http://shade.ki.ku.dk], and the improvement over the original SHADE results is substantial, suggesting that this is now the most readily and widely applicable of the three approximate procedures. Using this new library – SHADE2 – it is shown that, in line with expectations, a segmented rigid-body description of the heavy atoms yields only a small improvement in the agreement with neutron results. The SHADE2 library, now incorporated in the SHADE web server, is recommended as a routine procedure for deriving estimates of H-atom ADPs suitable for use in charge-density studies on molecular crystals, and its widespread use should reveal remaining deficiencies and perhaps overcome the inherent bias in the majority of such studies.

Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme to be characterised and provides evidence for bifunctionality of dCTP deaminase occurring outside the Archaea kingdom. A steady-state kinetic analysis revealed that the affinity for dCTP and deoxyuridine triphosphate as substrates for the synthesis of deoxyuridine monophosphate were very similar, a result that contrasts that obtained previously for the archaean Methanocaldococcus jannaschii enzyme, which showed approximately 10-fold lower affinity for deoxyuridine triphosphate than for dCTP. The crystal structures of the enzyme in complex with the inhibitor, thymidine triphosphate, and the apo form have been solved. Comparison of the two shows that upon binding of thymidine triphosphate, the disordered C-terminal arranges as a lid covering the active site, and the enzyme adapts an inactive conformation as a result of structural changes in the active site. In the inactive conformation dephosphorylation cannot take place due to the absence of a water molecule otherwise hydrogen-bonded to O2 of the α-phosphate.

The bacteriophage-encoded repressor protein plays a key role in determining the life cycle of a temperate phage following infection of a sensitive host. The repressor protein CI, which is encoded by the temperate lactococcal phage TP901-1, represses transcription from both the lytic promoter P(L) and the lysogenic promoter P(R) by binding to multiple operator sites on the DNA. In this study, we used a small bistable genetic switch element from phage TP901-1 to study the effect of cI deletions in vivo and showed that 43 amino acids could be removed from the C-terminal end of CI without destroying the ability of CI to repress transcription from the P(L) or the bistable switch properties. We showed that a helix-turn-helix motif located in the N-terminal part of CI is involved in DNA binding by introducing specific point mutations. Purification of CI and truncated forms of CI followed by analytical gel filtration and chemical cross-linking demonstrated that the C-terminal end of CI was required for oligomerization and that CI may exist as a hexamer in solution. Furthermore, expression and purification of the C-terminal part of CI (amino acids 92-180) showed that this part of the protein contained all the amino acids required to form an oligomer with an apparent molecular weight corresponding to a hexamer. We found that the C-terminal end of CI was required for de-repression of the P(L) following SOS induction, suggesting that the hexameric form of CI is needed for this or that this part of the protein is involved in the interaction with host proteins. By using small-angle X-ray scattering, we show for the first time the overall solution structure of a full-length wild-type bacteriophage repressor at low resolution revealing that the TP901-1 repressor forms a flat oligomer, most probably a trimer of dimers.

The trimeric dCTP deaminase produces dUTP that is hydrolysed to dUMP by the structurally closely related dUTPase. This pathway provides 70-80% of the total dUMP as a precursor for dTTP. Accordingly, dCTP deaminase is regulated by dTTP, which increases the substrate concentration for half-maximal activity and the cooperativity of dCTP saturation. Likewise, increasing concentrations of dCTP increase the cooperativity of dTTP inhibition. Previous structural studies showed that the complexes of inactive mutant protein, E138A, with dUTP or dCTP bound, and wild-type enzyme with dUTP bound were all highly similar and characterized by having an ordered C-terminal. When comparing with a new structure in which dTTP is bound to the active site of E138A, the region between Val120 and His125 was found to be in a new conformation. This and the previous conformation were mutually exclusive within the trimer. Also, the dCTP complex of the inactive H121A was found to have residues 120-125 in this new conformation, indicating that it renders the enzyme inactive. The C-terminal fold was found to be disordered for both new complexes. We suggest that the cooperative kinetics are imposed by a dTTP-dependent lag of product formation observed in presteady-state kinetics. This lag may be derived from a slow equilibration between an inactive and an active conformation of dCTP deaminase represented by the dTTP complex and the dUTP/dCTP complex, respectively. The dCTP deaminase then resembles a simple concerted system subjected to effector binding, but without the use of an allosteric site.

hDDPI (human dipeptidyl peptidase I) is a lysosomal cysteine protease involved in zymogen activation of granule-associated proteases, including granzymes A and B from cytotoxic T-lymphocytes and natural killer cells, cathepsin G and neutrophil elastase, and mast cell tryptase and chymase. In the present paper, we provide the first crystal structure of an hDPPI–inhibitor complex. The inhibitor Gly-Phe-CHN2 (Gly-Phe-diazomethane) was co-crystallized with hDPPI and the structure was determined at 2.0 Å (1 Å=0.1 nm) resolution. The structure of the native enzyme was also determined to 2.05 Å resolution to resolve apparent discrepancies between the complex structure and the previously published structure of the native enzyme. The new structure of the native enzyme is, within the experimental error, identical with the structure of the enzyme–inhibitor complex presented here. The inhibitor interacts with three subunits of hDPPI, and is covalently bound to Cys234 at the active site. The interaction between the totally conserved Asp1 of hDPPI and the ammonium group of the inhibitor forms an essential interaction that mimics enzyme–substrate interactions. The structure of the inhibitor complex provides an explanation of the substrate specificity of hDPPI, and gives a background for the design of new inhibitors.

The syntheses of the square-planar platinum(II) complexes [Pt(L)(tba)] (L = 5,5′-dichloro-2,2′-dihydroxyazobenzenate (dhab), (5-chloro-2-hydroxyphenylazo)-3-oxo-N-phenylbutanamidate (hpab) or (5-chloro-2-hydroxyphenylazo)-2-naphtholate (hpan) and (tba) = tributylamine) is reported, together with those of the complete set of analogous complexes [M(L)(py)] (M = nickel(II), palladium(II), platinum(II) and (py) = pyridine). The coordinating nitrogen has been assigned to the azo-nitrogen attached to the 5-chloro-2-hydroxyphenyl substituent in both [Ni(hpab)(py)] and [Pt(hpan)(tba)] by single crystal X-ray diffraction methods. It has been established that complexes with different group 10 metals are iso-structural by isomorphology of the crystals as determined by X-ray powder diffraction studies on the complexes containing pyridine in the fourth coordination site. Furthermore, we present a method to determine the coordination geometry by comparison of calculated 13C chemical shifts for possible coordination modes optimized by ab initio methods with experimentally measured 13C chemical shifts.

Tri-t-butylgallium has been reacted with the macrocycle 1,12-diaza-3,4:9,10-dibenzo-5,8-dioxocyclopentadecane which could be a potential ligand for gallium(III). A reaction product was analyzed and single crystal X-ray diffraction experiments showed that it consisted of the cyclic dimer of di-t-butylgallium-hydroxide hydrogen bonded to the macrocycle. Without a co-crystallized organic molecule di-t-butylgallium hydroxide crystallizes as a trimer. Density functional calculations have been used to predict the structures and the total energies for the monomer, dimer, trimer, and tetramer of dimethylgallium hydroxide in order to provide a basis for the understanding of oligomer population for the dimethylgallium hydroxides. Force field calculations are shown to be able to produce a similar strain energy difference for dimer, trimer, and tetramer forms of (CH3)2Ga(OH) and this method can economically be used for larger alkyl groups. The force field computations show that the trimeric di-t-butyl gallium(III) hydroxide is much more stable than the dimeric form which therefore must owe its existence to the association with the hydrogen-bonded macrocycle.

The upp gene, encoding uracil phosphoribosyltransferase (UPRTase) from the thermoacidophilic archaeon Sulfolobus solfataricus, was cloned and expressed in Escherichia coli. The enzyme was purified to homogeneity. It behaved as a tetramer in solution and showed optimal activity at pH 5.5 when assayed at 60 °C. Enzyme activity was strongly stimulated by GTP and inhibited by CTP. GTP caused an approximately 20-fold increase in the turnover number kcat and raised the Km values for 5-phosphoribosyl-1-diphosphate (PRPP) and uracil by two- and >10-fold, respectively. The inhibition by CTP was complex as it depended on the presence of the reaction product UMP. Neither CTP nor UMP were strong inhibitors of the enzyme, but when present in combination their inhibition was extremely powerful. Ligand binding analyses showed that GTP and PRPP bind cooperatively to the enzyme and that the inhibitors CTP and UMP can be bound simultaneously (KD equal to 2 and 0.5 µm, respectively). The binding of each of the inhibitors was incompatible with binding of PRPP or GTP. The data indicate that UPRTase undergoes a transition from a weakly active or inactive T-state, favored by binding of UMP and CTP, to an active R-state, favored by binding of GTP and PRPP.

dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg115 and Glu138, were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wild-type E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg2+, and the mutant enzyme also with the substrate dCTP and Mg2+. The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg2+ that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.

Uracil phosphoribosyltransferase (UPRTase) catalyzes the conversion of 5-phosphate-alpha-1-diphosphate (PRPP) and uracil to uridine 5'-monophosphate (UMP) and diphosphate. The UPRTase from Sulfolobus solfataricus has a unique regulation by nucleoside triphosphates compared to UPRTases from other organisms. To understand the allosteric regulation, crystal structures were determined for S. solfataricus UPRTase in complex with UMP and with UMP and the allosteric inhibitor CTP. Also, a structure with UMP bound in half of the active sites was determined. All three complexes form tetramers but reveal differences in the subunits and their relative arrangement. In the UPRTase-UMP complex, the peptide bond between a conserved arginine residue (Arg80) and the preceding residue (Leu79) adopts a cis conformation in half of the subunits and a trans conformation in the other half and the tetramer comprises two cis-trans dimers. In contrast, four identical subunits compose the UPRTase-UMP-CTP tetramer. CTP binding affects the conformation of Arg80, and the Arg80 conformation in the UPRTase-UMP-CTP complex leaves no room for binding of the substrate PRPP. The different conformations of Arg80 coupled to rearrangements in the quaternary structure imply that this residue plays a major role in regulation of the enzyme and in communication between subunits. The ribose ring of UMP adopts alternative conformations in the cis and trans subunits of the UPRTase-UMP tetramer with associated differences in the interactions of the catalytically important Asp209. The active-site differences have been related to proposed kinetic models and provide an explanation for the regulatory significance of the C-terminal Gly216.

Extensive and precise X-ray diffraction data for xylitol have been used to test different approaches to estimate nuclear parameters for H atoms in charge-density studies. The parameters from a neutron diffraction study of the same compound were taken as a reference. The resulting static charge densities obtained for the different approaches based on a multipole model were subjected to a topological analysis. The comparative analysis led to the following results. The procedure of extending the X-H bond to match bond lengths from neutron diffraction studies provides the best agreement with the neutron positional parameters. An isotropic model for the atomic displacements of H atoms is highly unsatisfactory and leads to significant deviations for the properties of the bond critical points including those that only involve non-H atoms. Anisotropic displacement parameters for H atoms can be derived from the X-ray data that are in agreement with the values from the neutron study, and the resulting charge-density models are in good agreement with the reference model. The anisotropic displacement parameters for H atoms are derived from the X-ray data as a sum of the external (rigid-body) and internal vibrations. The external vibrations are obtained from a TLS analysis of the ADPs of the non-H atoms and the internal vibrations from analysis of neutron diffraction studies of related compounds. The results from the analysis of positional and thermal parameters were combined to devise a 'best anisotropic' model, which was employed for three other systems where X-ray and neutron data were available. The results from the topological analysis of these systems confirm the success of the 'best anisotropic' model in providing parameters for the H atoms that give charge densities in agreement with the reference models based on H-atom parameters derived from neutron diffraction.

The zinc-containing cytidine deaminase (CDA, EC 3.5.4.5) is a pyrimidine salvage enzyme catalyzing the hydrolytic deamination of cytidine and 2'-deoxycytidine forming uridine and 2'-deoxyuridine, respectively. Homodimeric CDA (D-CDA) and homotetrameric CDA (T-CDA) both contain one zinc ion per subunit coordinated to the catalytic water molecule. The zinc ligands in D-CDA are one histidine and two cysteine residues, whereas in T-CDA zinc is coordinated to three cysteines. Two of the zinc coordinating cysteines in T-CDA form hydrogen bonds to the conserved residue Arg56, and this residue together with the dipole moments from two alpha-helices partially neutralizes the additional negative charge in the active site, leading to a catalytic activity similar to D-CDA. Arg56 has been substituted by a glutamine (R56Q), the corresponding residue in D-CDA, an alanine (R56A), and an aspartate (R56D). Moreover, one of the zinc-liganding cysteines has been substituted by histidine to mimic D-CDA, alone (C53H) and in combination with R56Q (C53H/R56Q). R56A, R56Q, and C53H/R56Q contain the same amount of zinc as the wild-type enzyme. The zinc-binding capacity of R56D is reduced. Only R56A, R56Q, and C53H/R56Q yielded measurable CDA activity, R56A and R56Q with similar K(m) but decreased V(max) values compared to wild-type enzyme. Because of dissociation into its inactive subunits, it was impossible to determine the kinetic parameters for C53H/R56Q. R56A and C53H/R56Q display increased apparent pK(a) values compared to the wild-type enzyme and R56Q. On the basis of the structures of R56A, R56Q, and C53H/R56Q an explanation is provided of kinetic results and the apparent instability of C53H/R56Q.

Rhamnogalacturonan lyase (RG-lyase) specifically recognizes and cleaves α-1,4 glycosidic bonds between l-rhamnose and d-galacturonic acids in the backbone of rhamnogalacturonan-I, a major component of the plant cell wall polysaccharide, pectin. The three-dimensional structure of RG-lyase from Aspergillus aculeatus has been determined to 1.5 Å resolution representing the first known structure from polysaccharide lyase family 4 and of an enzyme with this catalytic specificity. The 508-amino acid polypeptide displays a unique arrangement of three distinct modular domains. Each domain shows structural homology to non-catalytic domains from other carbohydrate active enzymes.

Dihydrorotate dehydrogenase (DHOD) (EC 1.3.99.11) catalyzes the fourth step and only redox reaction in the de novo pyrimidine biosynthesis, the stereospecific oxidation of (S)-DHO to orotate accompanied by the reduction of the prosthetic flavin (FMN) group (Fig. 1 ▶). A phylogenetic analysis of available DHOD sequences revealed that DHOD from different organisms can be assigned to two different major classes: class 1 and class 2 (Bjornberg et al. 1997). Class 1 DHODs originating mainly from gram-positive bacteria can furthermore be divided into subclasses 1A, 1B, and a new type 1S identified in Sulfolobus solfataricus (Sorensen and Dandanell 2002). The DHODs belonging to the different classes differ also in their location in the cell. The class 1A and 1B DHODs are found in the cytosol, whereas those from class 2 are membrane associated. Another distinct difference between the two classes of enzymes is their natural electron acceptor used to reoxidize the flavin group. Figure 1. The reaction catalyzed by class 2 DHODs and chemical structure of the DHOD inhibitors atovaquone, brequinar, and A771726. This figure is produced by ISIS draw 2.4 (MDL Information Systems, Inc.). Lactococcus lactis contains two genes encoding for DHODs representing subclass 1A and 1B, DHODA and DHODB, respectively. They differ in their structural organization and use of electron acceptor. The DHODA enzyme is a homodimer comprising two PyrDA subunits with an (αβ)8 barrel fold and the prosthetic FMN group located at the C-terminal ends of the β-strands at the top of the barrel (Rowland et al. 1997); it uses fumarate as its natural electron acceptor (Andersen et al. 1996). The DHODB is a heterotetramer composed of a central homodimer of PyrDB subunits resembling the DHODA structure and two PyrK subunits (Rowland et al. 2000). It is the presence of the PyrK subunits, which contain an FAD group and a [2Fe-2S] cluster, that enables the class 1B enzymes to use NAD+ as the natural electron acceptor (Nielsen et al. 1996). Class 1S DHOD can use Q0 and molecular oxygen as electron acceptors, together with the unphysiological substrates ferricyanide and DCIP used in in vitro measurements (Sorensen and Dandanell 2002). The membrane-associated class 2 DHODs found in gram-negative bacteria and in eukaryotes are monomeric enzymes that have the respiratory quinones as their physiological electron acceptors (Fig. 1 ▶; Bjornberg et al. 1999). A major structural difference between the class 1 and class 2 DHODs is their extended N terminus. The structure determinations for the DHODC and DHODH, truncated to be of the same length as DHODC, showed that the N terminus in the class 2 enzymes comprises a separate domain with two α-helices located on the top of the catalytic (αβ)8 barrel close to the FMN group (Liu et al. 2000; Norager et al. 2002). All eukaryotic enzymes from class 2 are located in the mitochondrial membrane attached by transmembrane α -helices, whereas the gram-negative bacterial enzymes are associated with the cytosolic side of the outer membrane. The extension of the N terminus in class 2 DHODs is thought to serve as a targeting signal guiding the enzyme to its location in the inner mitochondrial membrane (Rawls et al. 2000; Loffler et al. 2002) A basic residue in the active site mediates the stereospecific oxidation of (S)-DHO. It is a cysteine in the class 1 enzymes (Bjornberg et al. 1997) and a serine residue in the class 2 DHODs (Bjornberg et al. 1999). The basic residue is located in a loop in close contact to DHO bound on top of the FMN group. This position facilitates abstraction of a proton from the C5 atom of DHO in the enzymatic reaction, where a double bond between C5 and C6 is formed due to the transfer of a hydride ion from C6 to the N5 atom of FMN (Fig. 1 ▶). The second half reaction uses the respiratory quinones as electron acceptors. Their proposed binding site (Liu et al. 2000) is the N-terminal domain, where they are able to mediate the electron transfer to the FMNH2 group bound in the (αβ)8 barrel, as shown in Figure 1 ▶. The inhibition of DHODs causes a lowering of the intracellular pools of uracil, cytosine, and thymine nucleotides in cells, which makes DHODs attractive drug targets (Fairbanks et al. 1995). Most organisms are able to use a salvage pathway for pyrimidine nucleotide biosynthesis. It allows the pyrimidine bases or nucleosides formed from degradation of nucleotides and nucleic acids to be reused by salvage reactions. Some of the genes encoding for the enzymes in the pyrimidine salvage pathway were not identified in the genomes of two organisms affecting human health, the bacterium Helicobacter pylori causing stomach ulcers and stomach cancer and the malaria-causing parasite Plasmodium. They therefore depend exclusively on de novo synthesis of pyrimidine nucleotides, which explains why DHODs from these organisms are very attractive drug targets. Rapidly dividing human cells, like activated lymphocytes (Cutolo et al. 2003) and cancer cells (Shawver et al. 1997) require also a functional de novo nucleotide pathway to meet their requirement for nucleotides because recycling using salvage pathways of the already existing nucleotide pool through salvage pathways is not sufficient (Fairbanks et al. 1995). The immunomodulating drug leflunomide (Arava) has been approved for the treatment of rheumatoid arthritis (Goldenberg 1999). It has been shown that this drug inhibits DHODH and thereby inhibits the pyrimidine de novo biosynthetic pathway (Davis et al. 1996). The structure of DHODH is known in complex with A771726, the active metabolite of the prodrug leflunomide (DHODH-lefl) and brequinar (DHODH-breq) (Liu et al. 2000). From the analysis of the two structures of DHODH, it was concluded that the inhibitors could bind to the same site as the second natural substrate, the respiratory quinone. This feature was also deduced from enzyme kinetics studies of most of the class 2 enzyme inhibitors reported so far (Bader et al. 1998; Knecht et al. 2000). Considerable efforts have been put into structure activity analysis for DHODs from different organisms, among them rat and mouse (Knecht et al. 2000). An interesting feature of class 2 DHODs is the relatively small number of conserved residues located in their extended N termini. This explains why the N-terminal domains in the known structures of the class 2 DHODs display significant variations in the length and orientation of the helices that form this domain (Norager et al. 2002). The comparison of the sequences from DHODH, DHODC, and DHODR in Figure 2 ▶ reveals that, among the residues corresponding to the first 40 residues of DHODC, there are only six conserved. It is likely that this variation is the origin of the different behavior of inhibitors even for very closely related DHODs like the rat and human (Knecht and Loffler 1998). Thus, it seems possible to design inhibitors that are specific for a given organism, as demonstrated by structure-activity studies made on class 2 DHODs (Copeland et al. 2000). Figure 2. Structural alignment of DHODR, DHODH, and DHODC sequences. The structural elements correspond to the DHODR structures. α-Helixes in the central barrel are named α1–α8 and β-sheets in the barrel are named β1–β8. ... The work presented here addresses the differences between the class 2 DHODs. We have determined the crystal structures of the DHOD from rat, truncated like DHODH to be of the same length as DHODC, in complex with brequinar (DHODR-breq) and atovaquone (DHODR-ato). Atovaquone (Fig. 1 ▶) is a structural analog of ubiquinone. It is used as a broad-spectrum antiparasitic drug and has showed activity against various parasitic infections, such as malaria, toxoplasmosis (caused by Toxoplasma gondii), and pneumonia (Pneumocystis carinii) (Kaneshiro et al. 2000). Atovaquone has passed clinical trials and thereby received approval to combat Plasmodium falciparum. The primary mechanism of action in Plasmodium falciparum is the irreversible binding to the mitochondrial cytochrome bc1 complex, but it is also a potent inhibitor of DHOD activity (Ittarat et al. 1994). Atovaquone is marketed in the United States under the trade name Mepron. Atovaquone is one of the active compounds in Malarone (GlaxoSmithKline), used in the prophylaxis (prevention) and treatment of malaria. Brequinar is a quinoline carboxylic acid, which has been tested preclinically as a cytostatic agent. The three inhibitors atovaquone, A77126, and brequinar (known to inhibit different class 2 DHODs) are chemically different and do not mimic the natural electron acceptor, as shown in Figure 1 ▶. Our analysis of the two structures of DHODR-ato and DHODR-breq revealed a remarkable difference in the conformation of their small N-terminal domain. A comparison to the structures of inhibited DHODH have revealed subtle differences in the N-terminal domain that can explain why the DHOD inhibitors act differently on the two highly homologous enzymes. These results are valuable for the structural-based drug design of organism-specific inhibitors of DHOD, and have formed the basis for a modeling of quinone binding. Furthermore, we present an analysis of the differences in the N-terminal domain between the class 2 membrane-bound and membrane-associated DHOD, based on computational GRID modeling.

The glycoprotein rhamnogalacturonan acetylesterase from Aspergillus aculeatus has been crystallized in two crystal forms, an orthorhombic and a trigonal crystal form. In the orthorhombic crystal form, the covalently bound carbohydrate at one of the two N-glycosylation sites is involved in crystal contacts. The orthorhombic crystal form was obtained at pH 5.0 and the trigonal crystal form at pH 4.5. In one case, the two crystal forms were found in the same drop at pH 4.7. The differences in crystal packing in the two crystal forms can be explained by the pH-dependent variation in the protonation state of the glutamic acid residues on the protein surface.

The structure of the DNA-binding NAC domain of Arabidopsis ANAC (abscisic-acid-responsive NAC) has been determined by X-ray crystallography to 1.9A resolution (Protein Data Bank codes 1UT4 and 1UT7). This is the first structure determined for a member of the NAC family of plant-specific transcriptional regulators. NAC proteins are characterized by their conserved N-terminal NAC domains that can bind both DNA and other proteins. NAC proteins are involved in developmental processes, including formation of the shoot apical meristem, floral organs and lateral shoots, as well as in plant hormonal control and defence. The NAC domain does not possess a classical helix-turn-helix motif; instead it reveals a new transcription factor fold consisting of a twisted beta-sheet surrounded by a few helical elements. The functional dimer formed by the NAC domain was identified in the structure, which will serve as a structural template for understanding NAC protein function at the molecular level.

The bifunctional dCTP deaminase-dUTPase (DCD-DUT) from Methanocaldococcus jannaschii catalyzes the deamination of the cytosine moiety in dCTP and the hydrolysis of the triphosphate moiety forming dUMP, thereby preventing uracil from being incorporated into DNA. The crystal structure of DCD-DUT has been determined to 1.88-A resolution and represents the first known structure of an enzyme catalyzing dCTP deamination. The functional form of DCD-DUT is a homotrimer wherein the subunits are composed of a central distorted beta-barrel surrounded by two beta-sheets and four helices. The trimeric DCD-DUT shows structural similarity to trimeric dUTPases at the tertiary and quaternary levels. There are also additional structural elements in DCD-DUT compared with dUTPase because of a longer primary structure. Four of the five conserved sequence motifs that create the active sites in dUTPase are found in structurally equivalent positions in DCD-DUT. The last 25 C-terminal residues of the 204-residue-long DCD-DUT are not visible in the electron density map, but, analogous to dUTPases, the C terminus is probably ordered, closing the active site upon catalysis. Unlike other enzymes catalyzing the deamination of cytosine compounds, DCD-DUT is not exploiting an enzyme-bound metal ion such as zinc or iron for nucleophile generation. The active site contains two water molecules that are engaged in hydrogen bonds to the invariant residues Ser118, Arg122, Thr130, and Glu145. These water molecules are potential nucleophile candidates in the deamination reaction.

beta-1,4-Galactanases hydrolyze the galactan side chains that are part of the complex carbohydrate structure of the pectin. They are assigned to family 53 of the glycoside hydrolases and display significant variations in their pH and temperature optimum and stability. Two fungal beta-1,4-galactanases from Myceliophthora thermophila and Humicola insolens have been cloned and heterologously expressed, and the crystal structures of the gene products were determined. The structures are compared to the previously only known family 53 structure of the galactanase from Aspergillus aculeatus (AAGAL) showing approximately 56% identity. The M. thermophila and H. insolens galactanases are thermophilic enzymes and are most active at neutral to basic pH, whereas AAGAL is mesophilic and most active at acidic pH. The structure of the M. thermophila galactanase (MTGAL) was determined from crystals obtained with HEPES and TRIS buffers to 1.88 A and 2.14 A resolution, respectively. The structure of the H. insolens galactanase (HIGAL) was determined to 2.55 A resolution. The thermostability of MTGAL and HIGAL correlates with increase in the protein rigidity and electrostatic interactions, stabilization of the alpha-helices, and a tighter packing. An inspection of the active sites in the three enzymes identifies several amino acid substitutions that could explain the variation in pH optimum. Examination of the activity as a function of pH for the D182N mutant of AAGAL and the A90S/ H91D mutant of MTGAL showed that the difference in pH optimum between AAGAL and MTGAL is at least partially associated with differences in the nature of residues at positions 182, 90, and/or 91.

Seven amino-acid substitutions introduced into the 343 amino-acid-long sequence of Coprinus cinereus peroxidase (CiP) led to a mutant enzyme (TS-rCiP) which is more stable than the native enzyme at higher temperature, pH and hydrogen peroxide concentrations. It is therefore more suitable for industrial applications. A structure determination was conducted on a deglycosylated but still active form of TS-rCiP based on X-ray diffraction data to 2.05 A resolution measured on a crystal cooled to 100 K and refined to R = 0.202 and R(free) = 0.249. The increased stability of the TS-rCiP enzyme can be understood from the structural changes of the TS-rCiP structure revealed by a comparative analysis with other known CiP structures. One of the more significant changes caused by three of the substitutions, I49S, V53A and T121A, is the conversion of a hydrophobic pocket into a hydrophilic pocket with associated changes in the water structure and the hydrogen-bonding interactions. The E239G substitution, which gives rise to increased thermostability at high pH, creates changes in the water structure and in the orientation of a phenylalanine (Phe236) in its vicinity. The three substitutions M166F, M242 and Y242F introduced to increase the oxidative stability do not introduce any structural changes.

The influence of the different experimental parameters on the quality of the diffraction data collected on tetrafluoroterephthalonitrile (TFT) with a Nonius KappaCCD instrument has been examined. Data sets measured with different scan widths (0.25°, 0.50°, 1.0°) and scan times (70 s/° and 140 s/°) were compared with a highly redundant data set collected with an Enraf–Nonius CAD4 point detector diffractometer. As part of this analysis it was investigated how the parameters employed during the data reduction performed with theEvalCCDandSORTAVprograms affect the quality of the data. The KappaCCD data sets did not show any significant contamination from λ/2 radiation and possess good internal consistency with lowRintvalues. Decreasing the scan width seems to increase the standard uncertainties, which conversely are improved by an increase in the scan time. The suitability of the KappaCCD instrument to measure data to be used in charge density studies was also examined by performing a charge density data collection with the KappaCCD instrument. The same multipole model was used in the refinement of these data and of the CAD4 data. The two refinements gave almost identical parameters and residual electron densities. The topological analysis of the resulting static electron densities shows that the bond critical points have the same characteristics.

The preparation of the pentacyano(iminiumacetyl)cobaltate(III) anion and its N-methyl and N,N-dimethyl derivatives is reported. The iminiumacetyl group is formed by migratory insertion of cis hydrogen isocyanide in the pentacyano(methyl)cobaltate(III) anion. The new compounds have been spectroscopically characterized by (1)H, (13)C, (15)N, and (59)Co NMR spectroscopy and by absorption spectroscopy. The iminium carbon atoms yield (13)C NMR signals at 256.7, 247.7, and 240.4 ppm for the parent iminiumacetyl compound and its N-methyl and N,N-dimethyl derivatives, respectively. The (15)N resonance frequencies of the iminium groups and the lack of rotation of the carbon-nitrogen bond both show that this bond is best described as a double bond. The structure of (Et(4)N)(Ph(4)As)(2)[Co(CN)(5)(CH(3))] was determined by X-ray crystallography at 122.0(5) K. The structure displays disorder.

The flavoenzymes dihydroorotate dehydrogenases (DHODs) catalyze the fourth and only redox step in the de novo biosynthesis of UMP. Enzymes belonging to class 2, according to their amino acid sequence, are characterized by having a serine residue as the catalytic base and a longer N terminus. The structure of class 2 E. coli DHOD, determined by MAD phasing, showed that the N-terminal extension forms a separate domain. The catalytic serine residue has an environment differing from the equivalent cysteine in class 1 DHODs. Significant differences between the two classes of DHODs were identified by comparison of the E. coli DHOD with the other known DHOD structures, and differences with the class 2 human DHOD explain the variation in their inhibitors.

Recombinant rhamnogalacturonan lyase from Aspergillus aculeatus has been crystallized by a stepwise procedure and X-ray diffraction data have been collected. The crystals were grown using hanging-drop vapour-diffusion and microseeding techniques. Crystals were obtained showing a flat plate morphology. The crystallization conditions were 20% PEG 4000, 9% PEG 400, 0.1 M (NH(4))(2)SO(4) and 0.1 M sodium acetate pH 4.4. These crystals diffracted to a resolution of 1.5 A. The unit-cell parameters are a = b = 77.0, c = 170.8 A with the possible space group P4(3)2(1)2 or P4(1)2(1)2. There is most likely to be one molecule in the asymmetric unit, leading to a calculated solvent content of approximately 47% for the crystals.

The crystal structure of Thermoascus aurantiacus endoglucanase (Cel5A), a family 5 glycoside hydrolase, has been determined to 1.62 Å resolution by multiple isomorphous replacement with anomalous scattering. It is the first report of a structure in the subfamily to which Cel5A belongs. Cel5A consists solely of a catalytic module with compact eight-fold β/α barrel architecture. The length of the tryptophan-rich substrate binding groove suggests the presence of substrate binding subsites −4 to +3. Structural comparison shows that two glycines are completely conserved in the family, in addition to the two catalytic glutamates and six other conserved residues previously identified. Gly 44 in particular is part of a type IV C-terminal helix capping motif, whose disruption is likely to affect the position of an essential conserved arginine. One aromatic residue (Trp 170 in Cel5A), not conserved in term of sequence, is nonetheless spatially conserved in the substrate binding groove. Its role might be to force the bend that occurs in the polysaccharide chain on binding, thus favoring substrate distortion at subsite −1.

Uracil phosphoribosyltransferase (UPRTase) is part of the salvage pathway that leads to the biosynthesis of UMP. It catalyzes the formation of UMP and pyrophosphate from uracil and alpha-D-5-phosphoribosyl-1-pyrophosphate. Unlike enzymes in the de novo synthesis of UMP, UPRTases have only been found in lower organisms and are therefore potential targets for the development of new antibiotics. UPRTase from Bacillus caldolyticus has been crystallized and the structure has been determined by isomorphous replacement and refined to 3.0 A resolution. UPRTase from B. caldolyticus forms a dimer with the active sites pointing away from each other. A long arm from each subunit wraps around the other subunit, contributing half of the dimer interface. The monomer adopts the phosphoribosyltransferase type I fold, with a small C-terminal hood defining the uracil-binding site. The structure contains a well defined UMP molecule in the active site. The binding of UMP involves two sequence segments that are highly conserved among UPRTases. The first segment, Asp131-Ser139, contains the PRPP-binding consensus sequence motif known from other type I phosphoribosyltransferases and binds the ribose-5'-phosphate part of UMP. The second segment, Tyr193-Ala201, which is specific for uracil phosphoribosyltransferases, binds the uracil part of UMP through backbone contacts, partly mediated by a water molecule. Modelling of a PRPP-enzyme complex reveals that uracil can be activated to its tautomeric enol form by the complex. This is consistent with kinetic data, which display ordered sequential binding of substrates, with PRPP binding first. Based on this observation, a reaction mechanism is proposed.

Enzymatic coagulation of bovine milk can be divided in 2 steps: an enzymatic step, in which the Phe105-Met106 bond of the milk protein bovine κ-casein is cleaved, and an aggregation step. The aspartic peptidases bovine and camel chymosin (EC 3.4.23.4) are typically used to catalyze the enzymatic step. The most commonly used method to study chymosin activity is the relative milk-clotting activity test that measures the end point of the enzymatic and aggregation step. This method showed that camel chymosin has a 2-fold higher milk-clotting activity toward bovine milk than bovine chymosin. To enable a study of the enzymatic step independent of the aggregation step, a fluorescence resonance energy transfer assay has been developed using a peptide substrate derived from the 98–108 sequence of bovine κ-casein. This assay and Michaelis-Menten kinetics were employed to determine the enzymatic activity of camel and bovine chymosin under milk clotting-like conditions (pH 6.65, ionic strength 80 m M ). The results obtained show that the catalytic efficiency of camel chymosin is 3-fold higher than bovine chymosin. The substrate affinity and catalytic activity of bovine and camel chymosin increase at lower pH (6.00 and 5.50). The glycosylation of bovine and camel chymosin did not affect binding of the fluorescence resonance energy transfer substrate, but doubly glycosylated camel chymosin seems to have slightly higher catalytic efficiency. In the characterization of the enzymes, the developed assay is easier and faster to use than the traditionally used relative milk-clotting activity test method.

The nuclease domain of colicin E7 metallonuclease (NColE7) contains its active centre at the C-terminus. The mutant ΔN4-NColE7-C∗-where the four N-terminal residues including the positively charged K446, R447 and K449 are replaced with eight residues from the GST tag-is catalytically inactive. The crystal structure of this mutant demonstrates that its overall fold is very similar to that of the native NColE7 structure. This implicates the stabilizing effect of the remaining N-terminal sequence on the structure of the C-terminal catalytic site and the essential role of the deleted residues in the mechanism of the catalyzed reaction. Complementary QM/MM calculations on the protein-DNA complexes support the less favourable cleavage by the mutant protein than by NColE7. Furthermore, a water molecule as a possible ligand for the Zn2+-ion is proposed to play a role in the catalytic process. These results suggest that the mechanism of the Zn2+-containing HNH nucleases needs to be further studied and discussed.

The pyrG gene of Lactococcus lactis subsp. cremoris, encoding CTP synthase, has been cloned and sequenced. It is flanked upstream by an open reading frame showing homology to several aminotransferases and downstream by an open reading frame of unknown function. L. lactisstrains harboring disrupted pyrG alleles were constructed. These mutants required cytidine for growth, proving that in L. lactis, the pyrG product is the only enzyme responsible for the amination of UTP to CTP. In contrast to the situation in Escherichia coli, an L. lactis pyrG mutant could be constructed in the presence of a functionalcdd gene encoding cytidine deaminase. A characterization of the enzyme revealed similar properties as found for CTP synthases from other organisms. However, unlike the majority of CTP synthases the lactococcal enzyme can convert dUTP to dCTP, although a half saturation concentration of 0.6 mm for dUTP makes it unlikely that this reaction plays a significant physiological role. As for other CTP synthases, the oligomeric structure of the lactococcal enzyme was found to be a tetramer, but unlike most of the other previously characterized enzymes, the tetramer was very stable even at dilute enzyme concentrations.