The BRENDA enzyme information system-From a database to an expert system.

Enzymes, representing the largest and by far most complex group of proteins, play an essential role in all processes of life, including metabolism, gene expression, cell division, the immune system, and others. Their function, also connected to most diseases or stress control makes them interesting targets for research and applications in biotechnology, medical treatments, or diagnosis. Their functional parameters and other properties are collected, integrated, and made available to the scientific community in the BRaunschweig ENzyme DAtabase (BRENDA). In the last 30 years BRENDA has developed into one of the most highly used biological databases worldwide. The data contents, the process of data acquisition, data integration and control, the ways to access the data, and visualizations provided by the website are described and discussed.

BrEPS: a flexible and automatic protocol to compute enzyme-specific sequence profiles for functional annotation.

BACKGROUND: Models for the simulation of metabolic networks require the accurate prediction of enzyme function. Based on a genomic sequence, enzymatic functions of gene products are today mainly predicted by sequence database searching and operon analysis. Other methods can support these techniques: We have developed an automatic method "BrEPS" that creates highly specific sequence patterns for the functional annotation of enzymes. RESULTS: The enzymes in the UniprotKB are identified and their sequences compared against each other with BLAST. The enzymes are then clustered into a number of trees, where each tree node is associated with a set of EC-numbers. The enzyme sequences in the tree nodes are aligned with ClustalW. The conserved columns of the resulting multiple alignments are used to construct sequence patterns. In the last step, we verify the quality of the patterns by computing their specificity. Patterns with low specificity are omitted and recomputed further down in the tree. The final high-quality patterns can be used for functional annotation. We ran our protocol on a recent Swiss-Prot release and show statistics, as well as a comparison to PRIAM, a probabilistic method that is also specialized on the functional annotation of enzymes. We determine the amount of true positive annotations for five common microorganisms with data from BRENDA and AMENDA serving as standard of truth. BrEPS is almost on par with PRIAM, a fact which we discuss in the context of five manually investigated cases. CONCLUSIONS: Our protocol computes highly specific sequence patterns that can be used to support the functional annotation of enzymes. The main advantages of our method are that it is automatic and unsupervised, and quite fast once the patterns are evaluated. The results show that BrEPS can be a valuable addition to the reconstruction of metabolic networks.

Binding of inhibitory aromatic amino acids to Streptomyces griseus aminopeptidase.

The bacterial aminopeptidase isolated from the extracellular extract of Streptomyces griseus (SGAP) is a double-zinc exopeptidase with a high preference for large hydrophobic amino-terminus residues. It is a monomer of a relatively low molecular weight (30 kDa), is heat-stable, displays a high and efficient catalytic turnover and its activity is modulated by calcium ions. Several free amino acids were found to inhibit the activity of SGAP in the millimolar concentration range and can therefore serve for the study of binding of both inhibitors and reaction products. The current study is focused on the X-ray crystallographic analysis of the SGAP complexes with L-tryptophan and p-iodo-L-phenylalanine, both at 1.30 A resolution. These two bulky inhibitory amino acids were found to bind to the active site of SGAP in very similar positions and orientations. Both of them bind to the two active-site zinc ions via their free carboxylate group, while displacing the zinc-bound water/hydroxide that is present in the native enzyme. Further stabilization of the binding of the amino-acid carboxylate group is achieved by its relatively strong interactions with the hydroxyl group of Tyr246 and the carboxylate group of Glu131. The binding is also stabilized by three specific hydrogen bonds between the amine group of the bound amino acid and enzyme residues Glu131, Asp160 and Arg202. These consistent interactions confirm the key role of these residues in the specific binding of the free amine of substrates and products, as proposed previously. The phenyl ring of Phe219 of the enzyme is involved in stacking interactions with the corresponding aromatic ring of the bound affector. This interaction seems to be important for the binding and orientation of the aromatic side chain within the specificity pocket. These structural results correlate well with the results obtained for the complexes of SGAP with other inhibitory amino acids and support the general catalytic mechanism proposed for this and related enzymes.

The Helmholtz Network for Bioinformatics: an integrative web portal for bioinformatics resources.

SUMMARY: The Helmholtz Network for Bioinformatics (HNB) is a joint venture of eleven German bioinformatics research groups that offers convenient access to numerous bioinformatics resources through a single web portal. The 'Guided Solution Finder' which is available through the HNB portal helps users to locate the appropriate resources to answer their queries by employing a detailed, tree-like questionnaire. Furthermore, automated complex tool cascades ('tasks'), involving resources located on different servers, have been implemented, allowing users to perform comprehensive data analyses without the requirement of further manual intervention for data transfer and re-formatting. Currently, automated cascades for the analysis of regulatory DNA segments as well as for the prediction of protein functional properties are provided. AVAILABILITY: The HNB portal is available at http://www.hnbioinfo.de

Dynamic generation and qualitative analysis of metabolic pathways by a joint database/graph theoretical approach.

The dynamic generation and qualitative analysis of metabolic networks relying on continuously growing qualified metabolic data by a joint database/graph theoretical approach is described. The procedure is applied to analyze the connectivity of a metabolic network after enzyme removal and to subsequently perform shortest path analyses. The focus lies on the analysis of the connectivity of the metabolic network depending on model assumptions. Here we analyze the influence of the number of strongly connected components on the assignment of reversibility or irreversibility of the biochemical reactions.

ProClust: improved clustering of protein sequences with an extended graph-based approach.

MOTIVATION: The problem of finding remote homologues of a given protein sequence via alignment methods is not fully solved. In fact, the task seems to become more difficult with more data. As the size of the database increases, so does the noise level; the highest alignment scores due to random similarities increase and can be higher than the alignment score between true homologues. Comparing two sequences with an arbitrary alignment method yields a similarity value which may indicate an evolutionary relationship between them. A threshold value is usually chosen to distinguish between true homologue relationships and random similarities. To compensate for the higher probability of spurious hits in larger databases, this threshold is increased. Increasing specificity however leads to decreased sensitivity as a matter of principle. Sensitivity can be recovered by utilizing refined protocols. A number of approaches to this challenge have made use of the fact that proteins are often members of some larger protein family. This can be exploited by using position-specific substitution matrices or profiles, or by making use of transitivity of homology. Transitivity refers to the concept of concluding homology between proteins A and C based on homology between A and a third protein B and between B and C. It has been demonstrated that transitivity can lead to substantial improvement in recognition of remote homologues particularly in cases where the alignment score of A and C is below the noise level. A natural limit to the use of transitivity is imposed by domains. Domains, compact independent sub-units of proteins, are often shared between otherwise distinct proteins, and can cause substantial problems by incorrectly linking otherwise unrelated proteins. RESULTS: We extend a graph-based clustering algorithm which uses an asymmetric distance measure, scaling similarity values based on the length of the protein sequences compared. Additionally, the significance of alignment scores is taken into account and used for a filtering step in the algorithm. Post-processing, to merge further clusters based on profile HMMs is proposed. SCOP sequences and their super-family level classification are used as a test set for a clustering computed with our method for the joint data set containing both SCOP and SWISS-PROT. Note, the joint data set includes all multi-domain proteins, which contain the SCOP domains that are a potential source of incorrect links. Our method compares at high specificities very favorably with PSI-Blast, which is probably the most widely-used tool for finding remote homologues. We demonstrate that using transitivity with as many as twelve intermediate sequences is crucial to achieving this level of performance. Moreover, from analysis of false positives we conclude that our method seems to correctly bound the degree of transitivity used. This analysis also yields explicit guidance in choosing parameters. The heuristics of the asymmetric distance measure used neither solve the multi-domain problem from a theoretical point of view, nor do they avoid all types of problems we have observed in real data. Nevertheless, they do provide a substantial improvement over existing approaches. AVAILABILITY: The complete software source is freely available to all users under the GNU General Public License (GPL) from http://www.bioinformatik.uni-koeln.de/~proclust/download/

Clustering protein sequences--structure prediction by transitive homology.

MOTIVATION: It is widely believed that for two proteins Aand Ba sequence identity above some threshold implies structural similarity due to a common evolutionary ancestor. Since this is only a sufficient, but not a necessary condition for structural similarity, the question remains what other criteria can be used to identify remote homologues. Transitivity refers to the concept of deducing a structural similarity between proteins A and C from the existence of a third protein B, such that A and B as well as B and C are homologues, as ascertained if the sequence identity between A and B as well as that between B and C is above the aforementioned threshold. It is not fully understood if transitivity always holds and whether transitivity can be extended ad infinitum. RESULTS: We developed a graph-based clustering approach, where transitivity plays a crucial role. We determined all pair-wise similarities for the sequences in the SwissProt database using the Smith-Waterman local alignment algorithm. This data was transformed into a directed graph, where protein sequences constitute vertices. A directed edge was drawn from vertex A to vertex B if the sequences A and B showed similarity, scaled with respect to the self-similarity of A, above a fixed threshold. Transitivity was important in the clustering process, as intermediate sequences were used, limited though by the requirement of having directed paths in both directions between proteins linked over such sequences. The length dependency-implied by the self-similarity-of the scaling of the alignment scores appears to be an effective criterion to avoid clustering errors due to multi-domain proteins. To deal with the resulting large graphs we have developed an efficient library. Methods include the novel graph-based clustering algorithm capable of handling multi-domain proteins and cluster comparison algorithms. Structural Classification of Proteins (SCOP) was used as an evaluation data set for our method, yielding a 24% improvement over pair-wise comparisons in terms of detecting remote homologues. AVAILABILITY: The software is available to academic users on request from the authors. CONTACT: e.bolten@science-factory.com; schliep@zpr.uni-koeln.de; s.schneckener@science-factory.com; d.schomburg@uni-koeln.de; schrader@zpr.uni-koeln.de. SUPPLEMENTARY INFORMATION: http://www.zaik.uni-koeln.de/~schliep/ProtClust.html.

Interactions of Streptomyces griseus aminopeptidase with amino acid reaction products and their implications toward a catalytic mechanism.

Streptomyces griseus aminopeptidase (SGAP) is a double-zinc exopeptidase with a high preference toward large hydrophobic amino-terminus residues. It is a monomer of a relatively low molecular weight (30 kDa), it is heat stable, it displays a high and efficient catalytic turnover, and its activity is modulated by calcium ions. The small size, high activity, and heat stability make SGAP a very attractive enzyme for various biotechnological applications, among which is the processing of recombinant DNA proteins and fusion protein products. Several free amino acids, such as phenylalanine, leucine, and methionine, were found to act as weak inhibitors of SGAP and hence were chosen for structural studies. These inhibitors can potentially be regarded as product analogs because one of the products obtained in a normal enzymatic reaction is the cleaved amino terminal amino acid of the substrate. The current study includes the X-ray crystallographic analysis of the SGAP complexes with methionine (1.53 A resolution), leucine (1.70 A resolution), and phenylalanine (1.80 A resolution). These three high-resolution structures have been used to fully characterize the SGAP active site and to identify some of the functional groups of the enzyme that are involved in enzyme-substrate and enzyme-product interactions. A unique binding site for the terminal amine group of the substrate (including the side chains of Glu131 and Asp160, as well as the carbonyl group of Arg202) is indicated to play an important role in the binding and orientation of both the substrate and the product of the catalytic reaction. These studies also suggest that Glu131 and Tyr246 are directly involved in the catalytic mechanism of the enzyme. Both of these residues seem to be important for substrate binding and orientation, as well as the stabilization of the tetrahedral transition state of the enzyme-substrate complex. Glu131 is specifically suggested to function as a general base during catalysis by promoting the nucleophilic attack of the zinc-bound water/hydroxide on the substrate carbonyl carbon. The structures of the three SGAP complexes are compared with recent structures of three related aminopeptidases: Aeromonas proteolytica aminopeptidase (AAP), leucine aminopeptidase (LAP), and methionine aminopeptidase (MAP) and their complexes with corresponding inhibitors and analogs. These structural results have been used for the simulation of several species along the reaction coordinate and for the suggestion of a general scheme for the proteolytic reaction catalyzed by SGAP.

Crystallization and preliminary characterization of crystals of R-alcohol dehydrogenase from Lactobacillus brevis.

The R-specific alcohol dehydrogenase (RADH) from Lactobacillus brevis is a valuable catalyst for the production of chiral alcohols that can be used as synthons in asymmetric syntheses. RADH is a homotetramer with 222 symmetry and a molecular mass of 107 kDa. The recombinant enzyme has been expressed in Escherichia coli, purified to homogeneity and crystallized. The crystals belong to the orthorhombic space group I222, with unit-cell parameters a = 56.5, b = 85.1, c = 115.4 A, and diffract X-rays to at least 1.8 A resolution. The calculated crystal packing parameter V(M) = 2.59 A(3) Da(-1), corresponding to a solvent content of 52.5% and suggesting that one RADH monomer is contained in the asymmetric unit. The RADH tetramer lies on a special position with its molecular dyads coinciding with the crystallographic twofold axes and with its centre of mass on the origin of the unit cell.

Domain exchange: chimeras of Thermus aquaticus DNA polymerase, Escherichia coli DNA polymerase I and Thermotoga neapolitana DNA polymerase.

The intervening domain of the thermostable Thermus aquaticus DNA polymerase (TAQ: polymerase), which has no catalytic activity, has been exchanged for the 3'-5' exonuclease domain of the homologous mesophile Escherichia coli DNA polymerase I (E.coli pol I) and the homologous thermostable Thermotoga neapolitana DNA polymerase (TNE: polymerase). Three chimeric DNA polymerases have been constructed using the three-dimensional (3D) structure of the Klenow fragment of the E.coli pol I and 3D models of the intervening and polymerase domains of the TAQ: polymerase and the TNE: polymerase: chimera TaqEc1 (exchange of residues 292-423 from TAQ: polymerase for residues 327-519 of E.coli pol I), chimera TaqTne1 (exchange of residues 292-423 of TAQ: polymerase for residues 295-485 of TNE: polymerase) and chimera TaqTne2 (exchange of residues 292-448 of TAQ: polymerase for residues 295-510 of TNE: polymerase). The chimera TaqEc1 showed characteristics from both parental polymerases at an intermediate temperature of 50 degrees C: high polymerase activity, processivity, 3'-5' exonuclease activity and proof-reading function. In comparison, the chimeras TaqTne1 and TaqTne2 showed no significant 3'-5' exonuclease activity and no proof-reading function. The chimera TaqTne1 showed an optimum temperature at 60 degrees C, decreased polymerase activity compared with the TAQ: polymerase and reduced processivity. The chimera TaqTne2 showed high polymerase activity at 72 degrees C, processivity and less reduced thermostability compared with the chimera TaqTne1.

Crystallization, preliminary X-ray analysis of a native and selenomethionine D-hydantoinase from Thermus sp.

A D-hydantoinase from Thermus sp. was expressed in Escherichia coli, purified to homogeneity and crystallized both as native and Se-Met labelled protein. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 125.9, b = 215.8, c = 207.5 A. A three-wavelength MAD data set was collected to 2.5 A resolution and a native data set was collected to 1.7 A resolution. Crystal packing and self-rotation calculations led to the assumption of six protomers per asymmetric unit, corresponding to a V(M) value of 2.28 A(3) Da(-1) and a solvent content of 46%. As each protomer contains nine Se-Met residues, 54 selenium sites per asymmetric unit were present and could be unambigously located in the course of the MAD experiment. This selenium substructure is one of the largest selenium substructures that have been solved to date. The resulting phases obtained at a high-resolution limit of 3.0 A could be extended to 1.7 A and refined by application of density-modification techniques, especially non-crystallographic symmetry.

Identification of residues important both for primary receptor binding and specificity in fibroblast growth factor-7.

Fibroblast growth factors (FGFs) mediate a multitude of physiological and pathological processes by activating a family of tyrosine kinase receptors (FGFRs). Each FGFR binds to a unique subset of FGFs and ligand binding specificity is essential in regulating FGF activity. FGF-7 recognizes one FGFR isoform known as the FGFR2 IIIb isoform or keratinocyte growth factor receptor (KGFR), whereas FGF-2 binds well to FGFR1, FGFR2, and FGFR4 but interacts poorly with KGFR. Previously, mutations in FGF-2 identified a set of residues that are important for high affinity receptor binding, known as the primary receptor-binding site. FGF-7 contains this primary site as well as a region that restricts interaction with FGFR1. The sequences that confer on FGF-7 its specific binding to KGFR have not been identified. By utilizing domain swapping and site-directed mutagenesis we have found that the loop connecting the beta4-beta5 strands of FGF-7 contributes to high affinity receptor binding and is critical for KGFR recognition. Replacement of this loop with the homologous loop from FGF-2 dramatically reduced both the affinity of FGF-7 for KGFR and its biological potency but did not result in the ability to bind FGFR1. Point mutations in residues comprising this loop of FGF-7 reduced both binding affinity and biological potency. The reciprocal loop replacement mutant (FGF2-L4/7) retained FGF-2 like affinity for FGFR1 and for KGFR. Our results show that topologically similar regions in these two FGFs have different roles in regulating receptor binding specificity and suggest that specificity may require the concerted action of distinct regions of an FGF.

Mutation of recombinant catalytic subunit alpha of the protein kinase CK2 that affects catalytic efficiency and specificity.

In order to understand better the structural and functional relations between protein kinase CK2 catalytic subunit, the triphosphate moiety of ATP, the catalytic metal and the peptidic substrate, we built a structural model of Yarrowia lipolytica protein kinase CK2 catalytic subunit using the recently solved three-dimensional structure of the maize enzyme and the structure of cAMP-dependent protein kinase peptidic inhibitor (1CDK) as templates. The overall structure of the catalytic subunit is close to the structure solved by Niefind et al. It comprises two lobes, which move relative to each other. The peptide used as substrate is tightly bound to the enzyme, at specific locations. Molecular dynamic calculations in combination with the study of the structural model led us to identify amino acid residues close to the triphosphate moiety of ATP and a residue sufficiently far from the peptide that could be mutated so as to modify the specificity of the enzyme. Site-directed mutagenesis was used to replace by charged residues both glycine-48, a residue located within the glycine-rich loop, involved in binding of ATP phosphate moiety, and glycine-177, a residue close to the active site. Kinetic properties of purified wild-type and mutated subunits were studied with respect to ATP, MgCl(2) and protein kinase CK2 specific peptide substrates. The catalytic efficiency of the G48D mutant increased by factors of 4 for ATP and 17.5 for the RRRADDSDDDDD peptide. The mutant G48K had a low activity with ATP and no detectable activity with peptide substrates and was also inhibited by magnesium. An increased velocity of ADP release by G48D and the building of an electrostatic barrier between ATP and the peptidic substrate in G48K could explain these results. The kinetic properties of the mutant G177K with ATP were not affected, but the catalytic efficiency for the RRRADDSDDDDD substrate increased sixfold. Lysine 177 could interact with the lysine-rich cluster involved in the specificity of protein kinase CK2 towards acidic substrate, thereby increasing its activity.

Interactions of Streptomyces griseus aminopeptidase with a methionine product analogue: a structural study at 1.53 A resolution.

SGAP is an aminopeptidase present in the extracellular fluid of Streptomyces griseus cultures. It is a double-zinc enzyme with a strong preference for large hydrophobic amino-terminus residues. It is a monomeric (30 kDa) heat-stable enzyme, with a high and efficient catalytic activity modulated by calcium ions. The small size, high activity and heat stability make SGAP a very attractive enzyme for various biotechnological applications. Only one other related aminopeptidase (Aeromonas proteolytica AP; AAP) has been structurally analyzed to date and its structure was shown to be considerably similar to SGAP, despite the low sequence homology between the two enzymes. The motivation for the detailed structural analysis of SGAP originated from a strong mechanistic interest in the family of double-zinc aminopeptidases, combined with the high potential applicability of these enzymes. The 1.75 A crystallographic structure of native SGAP has been previously reported, but did not allow critical mechanistic interpretations owing to inconclusive structural regions around the active site. A more accurate structure of SGAP at 1.58 A resolution is reported in this paper, along with the 1.53 A resolution structure of the SGAP complex with inhibitory methionine, which is also a product of the SGAP catalytic process. These two high-resolution structures enable a better understanding of the SGAP binding mode of both substrates and products. These studies allowed the tracing of the previously disordered region of the enzyme (Glu196-Arg202) and the identification of some of the functional groups of the enzyme that are involved in enzyme-substrate interactions (Asp160, Met161, Gly201, Arg202 and Phe219). These studies also suggest that Glu131 is directly involved in the catalytic mechanism of SGAP, probably as the hydrolytic nucleophile. The structural results are compared with a recent structure of AAP with an hydroxamate inhibitor in order to draw general functional conclusions which are relevant for this family of low molecular-weight aminopeptidases.

Conserved arginine-516 of Penicillium amagasakiense glucose oxidase is essential for the efficient binding of beta-D-glucose.

The effects of mutation of key conserved active-site residues (Tyr-73, Phe-418, Trp-430, Arg-516, Asn-518, His-520 and His-563) of glucose oxidase from Penicillium amagasakiense on substrate binding were investigated. Kinetic studies on the oxidation of beta-D-glucose combined with molecular modelling showed the side chain of Arg-516, which forms two hydrogen bonds with the 3-OH group of beta-D-glucose, to be absolutely essential for the efficient binding of beta-D-glucose. The R516K variant, whose side chain forms only one hydrogen bond with the 3-OH group of beta-D-glucose, exhibits an 80-fold higher apparent K(m) (513 mM) but a V(max) only 70% lower (280 units/mg) than the wild type. The complete elimination of a hydrogen-bond interaction between residue 516 and the 3-OH group of beta-D-glucose through the substitution R516Q effected a 120-fold increase in the apparent K(m) for glucose (to 733 mM) and a decrease in the V(max) to 1/30 (33 units/mg). None of the other substitutions, with the exception of variant F418A, affected the apparent K(m) more than 6-fold. In contrast, the removal of aromatic or bulky residues at positions 73, 418 or 430 resulted in decreases in the maximum rates of glucose oxidation to less than 1/90. Variants of the potentially catalytically active His-520 and His-563 were completely, or almost completely, inactive. Thus, of the residues forming the active site of glucose oxidase, Arg-516 is the most critical amino acid for the efficient binding of beta-D-glucose by the enzyme, whereas aromatic residues at positions 73, 418 and 430 are important for the correct orientation and maximal velocity of glucose oxidation.

Purification of a D-hydantoinase using a laboratory-scale streamline phenyl column as the initial step.

A D-hydantoinase from Thermus sp. was overexpressed in Escherichia coli and purified to homogeneity for subsequent crystallization. The purification was performed with hydrophobic interaction chromatography as the capture step followed by anion-exchange chromatography and gel permeation chromatography as intermediate purification and polishing steps, respectively. The hydrophobic interaction step was done in fluidized bed mode in a laboratory-scale Streamline column made from conventional laboratory equipment. The whole purification protocol could be finished within one day. The purified enzyme crystallizes. The crystals are suitable for X-ray protein structure analysis and diffract to at least 2.3 A resolution. Complete data sets have been measured up to 2.6 A resolution. The X-ray structure is currently being solved.

GTP plus water mimic ATP in the active site of protein kinase CK2.

The structures of the catalytic subunit of protein kinase CK2 from Zea mays complexed with Mg2+ and with analogs of ATP or GTP were determined to 2.2 A resolution. Unlike most other protein kinases, CK2 from various sources shows 'dual-cosubstrate specificity', that is, the ability to efficiently use either ATP or GTP as a cosubstrate. The structures of these complexes demonstrate that water molecules are critical to switch the active site of CK2 from an ATP- to a GTP-compatible state. An understanding of the structural basis of dual-cosubstrate specificity may help in the design of drugs that target CK2 or other kinases with this property.

X-ray structure determination of a vanadium-dependent haloperoxidase from Ascophyllum nodosum at 2.0 A resolution.

The homo-dimeric structure of a vanadium-dependent haloperoxidase (V-BPO) from the brown alga Ascophyllum nodosum (EC 1.1.11.X) has been solved by single isomorphous replacement anomalous scattering (SIRAS) X-ray crystallography at 2.0 A resolution (PDB accession code 1QI9), using two heavy-atom datasets of a tungstate derivative measured at two different wavelengths. The protein sequence (SwissProt entry code P81701) of V-BPO was established by combining results from protein and DNA sequencing, and electron density interpretation. The enzyme has nearly an all-helical structure, with two four-helix bundles and only three small beta-sheets. The holoenzyme contains trigonal-bipyramidal coordinated vanadium atoms at its two active centres. Structural similarity to the only other structurally characterized vanadium-dependent chloroperoxidase (V-CPO) from Curvularia inaequalis exists in the vicinity of the active site and to a lesser extent in the central four-helix bundle. Despite the low sequence and structural similarity between V-BPO and V-CPO, the vanadium binding centres are highly conserved on the N-terminal side of an alpha-helix and include the proposed catalytic histidine residue (His418(V-BPO)/His404(V-CPO)). The V-BPO structure contains, in addition, a second histidine near the active site (His411(V-BPO)), which can alter the redox potential of the catalytically active VO2-O2 species by protonation/deprotonation reactions. Specific binding sites for the organic substrates, like indoles and monochlordimedone, or for halide ions are not visible in the V-BPO structure. A reaction mechanism for the enzymatic oxidation of halides is discussed, based on the present structural, spectroscopic and biochemical knowledge of vanadium-dependent haloperoxidases, explaining the observed enzymatic differences between both enzymes.

Mutations uncouple human fibroblast growth factor (FGF)-7 biological activity and receptor binding and support broad specificity in the secondary receptor binding site of FGFs.

The fibroblast growth factor (FGF) family plays a key role in a multitude of physiological and pathological processes. The activities of FGFs are mediated by a family of tyrosine kinase receptors, designated FGFRs. The mechanism by which FGFs induce receptor activation is controversial. Despite their structural similarity, FGFs display distinct receptor binding characteristics and cell type specificity. Previous studies with FGF-2 identified a low affinity receptor binding site that is located within a loop connecting its 9th and 10th beta-strands. The corresponding residues in the other family members are highly variable, and it was proposed that the variability might confer on FGFs unique receptor binding characteristics. We studied the role of this loop in FGF-7 by both site-directed mutagenesis and loop replacement. Unlike the other members of the FGF family, FGF-7 recognizes only one FGFR isoform and is, therefore, ideal for studies of how the specificity in the FGF-FGFR interaction is conferred at the structural level. Point mutations in the loop of FGF-7 did not change receptor binding affinity but resulted in reduced mitogenic potency and reduced ability to induce receptor-mediated phosphorylation events. These results suggest that the loop of FGF-7 fulfills the role of low affinity binding site required for receptor activation. The observation that it is possible to uncouple FGF-7 receptor binding and biological activity favors a bivalent model for FGFR dimerization, and it may be clinically relevant to the design of FGF-7 antagonists. Reciprocal loop replacement between FGF-7 and FGF-2 had no effect on their known receptor binding affinities nor did it alter their known specificity in eliciting a mitogenic response. In conclusion, these results suggest that, despite the diversity in the loop structure of FGF-2 and FGF-7, the loop has a similar function in both growth factors.

Identification of amino acids responsible for the oxygen sensitivity of ferredoxins from Anabaena variabilis using site-directed mutagenesis.

The filamentous cyanobacterium Anabaena variabilis (ATCC 29413) possesses two molybdenum dependent nitrogenase systems, nif1 and nif2. The nif1 system is regulated by a developmental program involving heterocyst differentiation; the nif2 system is expressed in all cells only under anaerobic conditions and the expression is controlled environmentally. The genes fdxH1 and fdxH2, encoding two [2Fe-2S] ferredoxins, are part of the these two distinct and differently regulated nif gene clusters. The sensitivity of both ferredoxins to oxygen was different; the half-life of FdxH2 in air was only approximately 1.5 h, while FdxH1 retained 80% of its nitrogenase activity after 24 h. We used site-directed mutagenesis to identify the role of individual amino acid residues responsible for oxygen sensitivity and found out that the FdxH2 double mutant I76A/V77L was much more resistant to oxygen than the wild-type ferredoxin (FdxH2) and similar to FdxH1. By modelling it was shown that the accessibility of the cavity around the iron-sulfur cluster was responsible for that.