Armando Negri

Protein HU binds specifically to kinked DNA

We have purified the main four‐way junction DNA‐binding protein of Escherichia coli, and have found It to be the well‐known HU protein. HU protein recognizes with high‐affinity one of the angles present in the junction, a molecule with the shape of an X. Other DNA structures characterized by sharp bends or kinks, like bulged duplex DNAs containing unpaired bases, are also bound. HU protein appears to inhibit cruciform extrusion from supercoiled inverted repeat (palindromic) DNA, either by constraining supercoiling or by trapping a metastable interconversion intermediate. All these properties are analogous to the properties of the mammalian chromatin protein HMG1. We suggest that HU is a prokaryotic HMG1‐like protein rather than a histone‐like protein.

Structure ofL-aspartate oxidase: implications for the succinate dehydrogenase/fumarate reductase oxidoreductase family

BACKGROUND Given the vital role of NAD+ in cell metabolism, the enzymes involved in bacterial de novo NAD+ biosynthesis are possible targets for drug design against pathogenic bacteria. The first reaction in the pathway is catalysed by L-aspartate oxidase (LASPO), a flavoenzyme that converts aspartate to iminoaspartate using either molecular oxygen or fumarate as electron acceptors. LASPO has considerable sequence homology with the flavoprotein subunits of succinate dehydrogenase (SDH) and fumarate reductase (FRD). RESULTS The crystal structure of the apoform of LASPO from Escherichia coli has been determined to 2.2 A resolution. The enzyme shows a novel fold for an FAD-dependent protein, comprising a three-domain structure: an FAD-binding domain with the dinucleotide-binding fold, a C-terminal three-helical bundle domain, and an alpha + beta capping domain, which is topologically similar to the small subunit of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase. The interface between the FAD-binding and capping domains defines a cleft in which the active site is located. CONCLUSIONS A number of strictly conserved residues present in all three domains indicate that LASPO, SDH and FRD share the same overall folding topology. Many of these conserved residues are in the FAD-binding site and active centre, suggesting a similar catalytic mechanism. Thus, LASPO, SDH and FRD form a class of functionally and structurally related oxidoreductases that are all able to reduce fumarate and to oxidise a dicarboxylate substrate.

Isolation of the epithiospecifier protein from oil-rape (Brassica napus ssp. oleifera) seed and its characterization.

The epithiospecifier protein (ESP) is a myrosinase (MYR) cofactor, which is necessary to drive the MYR‐catalyzed hydrolysis of some specific glucosinolates towards the production of cyanoepithioalkanes instead of isothiocyanates and nitriles. ESP was isolated from Brassica napus seeds by anionic exchange and gel filtration chromatography. ESP showed a molecular weight of about 39 kDa and pI 5.3. The amino acid sequence of several tryptic peptides of ESP (accounting for about 50% of the total sequence) made it possible to establish the high similarity (81% identity) with a hypothetical 37 kDa protein (TrEMBL data base accession number Q39104) and several jasmonate‐inducible proteins from Arabidopsis thaliana. This observation suggests that ESP is likely to be involved in jasmonate‐mediated defence and disease resistance mechanisms.

The primary structure of UK 114 tumor antigen

UK114 is a tumor antigen expressed by various malignant neoplasms. The complete amino acid sequence of UK114 purified from goat liver has been determined by automated Edman degradation of CNBr and endoproteinase Lys‐C peptides. The protein contains 137 amino acid residues, which corresponds to a molecular mass of 14 229 Da. MALDITOF analysis resulted in a molecular weight of 14 290, suggesting that the N‐terminal Met residue is acetylated. Sequence comparison shows that UK114 from goat liver (1) has 77% identity with a previously described 23 kDa protein from rat liver (Levy‐Favatier et al. (1993) Eur. J. Biochem. 212, 665–673), (2) shares a very high degree of similarity with a family of prokaryotic and eukaryotic hypothetic proteins whose function have not yet been characterized, and (3) exhibits a significant similarity to a group of tumor‐associated antigens which belongs to a superfamily of heat shock proteins, acting as possible targets for the hosts antitumor immunity.

Identification and characterization of a Bowman–Birk inhibitor active towards trypsin but not chymotrypsin in Lupinus albus seeds

The paper describes the purification, structural characterization and inhibitory properties of a trypsin inhibitor from Lupinus albus L., a leguminous plant believed to be devoid of any protease inhibitor. The protein has been isolated by a newly set-up procedure and characterized by direct amino acid sequencing, MALDI-TOF mass spectroscopy and circular dichroism. Inhibitory properties toward bovine trypsin and chymotrypsin, as well as its thermal and pH stabilities, have been also assessed. The inhibitor is 63 amino acid long (Mr 6858; pI 8.22) and it is capable to inhibit two trypsin molecules simultaneously, with a Kd of 4.2+/-0.4 nM, but not chymotrypsin. BLAST search against UniProtKB/TrEMBL database indicates that the inhibitor belongs to the Bowman-Birk inhibitor (BBI) family. The interest in these serine-protease inhibitors arises from the ability to prevent or suppress carcinogen-induced transformation, as shown in various in vitro and in vivo model systems.

Properties of the flavoenzyme D-aspartate oxidase from Octopus vulgaris.

The properties of D-aspartate oxidase from Octopus vulgaris (EC 1.4.3.1) have been investigated. The protein is a monomer of M(r) 37,000 containing one mol flavin/mol protein. The enzyme as isolated exists at least in two forms, one containing FAD and the other, which is catalytically inactive, probably containing 6-OH-FAD, as inferred from the absorption spectrum of the enzyme. An additional form of the enzyme, as far as the nature of the coenzyme is concerned, has been detected in the purified enzyme and shown to derive from the form originally containing FAD. The modulation of the coenzyme reactivity exerted by Octopus D-aspartate oxidase, as studied by spectrophotometric techniques, conforms to the one expected for an enzyme belonging to the oxidase class of flavoproteins. Structural investigations show similarities in both the amino-acid composition and the N-terminal amino-acid sequence to bovine D-aspartate oxidase and porcine D-amino-acid oxidase. In summary, the general properties of the enzyme from Octopus vulgaris closely resemble those of the enzyme from beef kidney. Moreover, kinetic analyses suggest that two active-site residues with pKa of 7.1 and 9.1 are critical for catalysis, and that the ionization of such residues has different effects on the catalytic activity depending whether mono- or dicarboxylic D-amino acids are used as substrate.

Purification of beef kidney d-aspartate oxidase overexpressed in Escherichia coli and characterization of its redox potentials and oxidative activity towards agonists and antagonists of excitatory amino acid receptors

The flavoenzyme d-aspartate oxidase from beef kidney (DASPO, EC 1.4. 3.1) has been overexpressed in Escherichia coli. A purification procedure, faster than the one used for the enzyme from the natural source (bDASPO), has been set up yielding about 2 mg of pure recombinant protein (rDASPO) per each gram of wet E. coli paste. rDASPO has been shown to possess the same general biochemical properties of bDASPO, except that the former contains only FAD, while the latter is a mixture of two forms, one active containing FAD and one inactive containing 6-OH-FAD (9-20% depending on the preparation). This results in a slightly higher specific activity (about 15%) for rDASPO compared to bDASPO and in facilitated procedures for apoprotein preparation and reconstitution. Redox potentials of -97 mV and -157 mV were determined for free and l-(+)-tartrate complexed DASPO, respectively, in 0.1 M KPi, pH 7.0, 25 degrees C. The large positive shift in the redox potential of the coenzyme compared to free FAD (-207 mV) is in agreement with similar results obtained with other flavooxidases. rDASPO has been used to assess a possible oxidative activity of the enzyme towards a number of compounds used as agonists or antagonists of neurotransmitters, including d-aspartatic acid, d-glutamic acid, N-methyl-d-aspartic acid, d,l-cysteic acid, d-homocysteic acid, d, l-2-amino-3-phosphonopropanoic acid, d-alpha-aminoadipic acid, d-aspartic acid-beta-hydroxamate, glycyl-d-aspartic acid and cis-2, 3-piperidine dicarboxylic acid. Kinetic parameters for each substrate in 50 mM KPi, pH 7.4, 25 degrees C are reported.

Characterization of l-aspartate oxidase and quinolinate synthase from Bacillus subtilis

NAD is an important cofactor and essential molecule in all living organisms. In many eubacteria, including several pathogens, the first two steps in the de novo synthesis of NAD are catalyzed by l‐aspartate oxidase (NadB) and quinolinate synthase (NadA). Despite the important role played by these two enzymes in NAD metabolism, many of their biochemical and structural properties are still largely unknown. In the present study, we cloned, overexpressed and characterized NadA and NadB from Bacillus subtilis, one of the best studied bacteria and a model organism for low‐GC Gram‐positive bacteria. Our data demonstrated that NadA from B. subtilis possesses a [4Fe–4S]2+ cluster, and we also identified the cysteine residues involved in the cluster binding. The [4Fe–4S]2+ cluster is coordinated by three cysteine residues (Cys110, Cys230, and Cys320) that are conserved in all the NadA sequences reported so far, suggesting a new noncanonical binding motif that, on the basis of sequence alignment studies, may be common to other quinolinate synthases from different organisms. Moreover, for the first time, it was shown that the interaction between NadA and NadB is not species‐specific between B. subtilis and Escherichia coli.

Inhibitory properties and solution structure of a potent Bowman-Birk protease inhibitor from lentil (Lens culinaris, L) seeds.

Bowman–Birk serine protease inhibitors are a family of small plant proteins, whose physiological role has not been ascertained as yet, while chemopreventive anticarcinogenic properties have repeatedly been claimed. In this work we present data on the isolation of a lentil (Lens culinaris, L., var. Macrosperma) seed trypsin inhibitor (LCTI) and its functional and structural characterization. LCTI is a 7448 Da double‐headed trypsin/chymotrypsin inhibitor with dissociation constants equal to 0.54 nm and 7.25 nm for the two proteases, respectively. The inhibitor is, however, hydrolysed by trypsin in a few minutes timescale, leading to a dramatic loss of its affinity for the enzyme. This is due to a substantial difference in the kon and k*on values (1.1 µm−1·s−1 vs. 0.002 µm−1·s−1), respectively, for the intact and modified inhibitor. A similar behaviour was not observed with chymotrypsin. The twenty best NMR structures concurrently showed a canonical Bowman–Birk inhibitor (BBI) conformation with two antipodal β‐hairpins containing the inhibitory domains. The tertiary structure is stabilized by ion pairs and hydrogen bonds involving the side chain and backbone of Asp10‐Asp26‐Arg28 and Asp36‐Asp52 residues. At physiological pH, the final structure results in an asymmetric distribution of opposite charges with a negative electrostatic potential, centred on the C‐terminus, and a highly positive potential, surrounding the antitryptic domain. The segment 53–55 lacks the anchoring capacity found in analogous BBIs, thus rendering the protein susceptible to hydrolysis. The inhibitory properties of LCTI, related to the simultaneous presence of two key amino acids (Gln18 and His54), render the molecule unusual within the natural Bowman–Birk inhibitor family.

Synthesis of Multifunctional PAMAM−Aminoglycoside Conjugates with Enhanced Transfection Efficiency

The development of multifunctional vectors for efficient and safe gene delivery is one of the major challenges for scientists working in the gene therapy field. In this context, we have designed a novel type of aminoglycoside-rich dendrimers with a defined structure based on polyamidoamine (PAMAM) in order to develop efficient, nontoxic gene delivery vehicles. Three different conjugates, i.e., PAMAM G4-neamine, -paromomycin, and -neomycin, were synthesized and characterized by nuclear magnetic resonance (NMR) and MALDI analysis. The conjugates were found to self-assemble electrostatically with plasmid DNA, and unlike neamine conjugate, each at its optimum showed increased gene delivery potency compared to PAMAM G4 dendrimer in three different cell lines, along with negligible cytotoxicity. These results all disclosed aminoglycosides as suitable functionalities for tailoring safe and efficient multifunctional gene delivery vectors.