Laccases and other four-copper oxidases are usually constructed of three domains: Domains one and three house the copper sites, and the second domain often helps form a substrate-binding cleft. In contrast to this arrangement, the genome of Streptomyces coelicolor was found to encode a small, four-copper oxidase that lacks the second domain. This protein is representative of a new family of enzymes-the two-domain laccases. Disruption of the corresponding gene abrogates laccase activity in the growth media. We have recombinantly expressed this enzyme, called SLAC, in Escherichia coli and characterized it. The enzyme binds four copper ions/monomer, and UV-visible absorption and EPR measurements confirm that the conserved type 1 copper site and trinuclear cluster are intact. We also report the first known paramagnetic NMR spectrum for the trinuclear copper cluster of a protein from the laccase family. The enzyme is highly stable, retaining activity as a dimer in denaturing gels after boiling and SDS treatment. The activity of the enzyme against 2,6-dimethoxyphenol (DMP) peaks at an unprecedentedly high pH (9.4), whereas the activity against ferrocyanide decreases with pH. SLAC binds negatively charged substrates more tightly than positively charged or uncharged molecules.Keywords: laccase; NMR; activity; multicopper; oxidase; copper; PPO The laccase class of enzymes is widely distributed throughout nature and exhibits a wide range of roles and substrate specificity. They are sometimes also referred to as polyphenol oxidases (PPOs) and are a part of the multi-copper oxidase family (Messerschmidt 1997). The first laccase was discovered in the tree Rhus vernicifera more than a hundred years ago (Yoshida 1883). The plant and fungal laccases are similar in that they have low substrate specificity, reacting with a host of derivatized phenols with K m of a few millimolar. The lack of substrate specificity often leaves the natural role of laccases in question, but this versatility has allowed new roles to be found for many in several commercial processes, including delignification, decolorization of industrial waste, and wine clarification (Mayer and Staples 2002).In contrast, ceruloplasmin (from the plasma of vertebrates) and Fet3p (from Saccharomyces cerevisiae) bind and oxidize Fe(II) specifically. CueO and PcoA are involved with metal homeostasis in Escherichia coli . Ascorbate oxidase, found in many plants, uses ascorbate as a substrate, but for unknown purposes. In this article, all of these proteins will be denoted laccases.With the recent advent of available genomic data, these enzymes are being isolated from a wide range of organisms, with a large increase in the number of enzymes originating from prokaryotic sources (Claus 2003). Highly conserved features of these proteins include a copper ion bound in a type 1, or blue, site and three more copper ions bound in a trinuclear cluster that is sometimes described as a sum of type 2 and type 3 sites. The polypeptide architecture typically consists of three cup...

Bacillus pasteurii UreG, a chaperone involved in the urease active site assembly, was overexpressed in Escherichia coli BL21(DE3) and purified to homogeneity. The identity of the recombinant protein was confirmed by SDS-PAGE, protein sequencing, and mass spectrometry. A combination of size exclusion chromatography and multiangle and dynamic laser light scattering established that BpUreG is present in solution as a dimer. Analysis of circular dichroism spectra indicated that the protein contains large portions of helices (15%) and strands (29%), whereas NMR spectroscopy indicated the presence of conformational fluxionality of the protein backbone in solution. BpUreG catalyzes the hydrolysis of GTP with a k cat ‫؍‬ 0.04 min ؊1 , confirming a role for this class of proteins in coupling energy requirements and nickel incorporation into the urease active site. BpUreG binds two Zn 2؉ ions per dimer, with a K D ‫؍‬ 42 ؎ 3 M, and has a 10-fold lower affinity for Ni 2؉ . A structural model for BpUreG was calculated by using threading algorithms. The protein, in the fully folded state, features the typical structural architecture of GTPases, with an open ␤-barrel surrounded by ␣-helices and a P-loop at the N terminus. The protein dynamic behavior observed in solution is critically discussed relative to the structural model, using algorithms for disorder predictions. The results suggest that UreG proteins belong to the class of intrinsically unstructured proteins that need the interaction with cofactors or other protein partners to perform their function. It is also proposed that metal ions such as Zn 2؉ could have important structural roles in the urease activation process.

The aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors. The protein contains six copper atoms per monomer, arranged in two centers named Cu(A) and Cu(Z). Cu(Z) could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)=2.015, A(x)=1.5 mT, g(y)=2.071, A(y)=2 mT, g(z)=2.138, A(z)=7 mT) and a strong absorption at approximately 640 nm. Cu(A) can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x)=g(y)= 2.021, A(x) = A(y)=0 mT, g(z) = 2.178, A(z)= 4 mT) and absorption bands at 480, 540, and approximately 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the Cu(A) center. In form A, Cu(A) is predominantly oxidized (S = (1)/(2), Cu(1.5+)-Cu(1.5+)), while in form B it is mostly in the one-electron reduced state (S = 0, Cu(1+)-Cu(1+)). In both forms, Cu(Z) remains reduced (S = 1/2). Complete crystallographic data at 2.4 A indicate that Cu(A) is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu(Z) is a novel tetracopper cluster [Brown, K., et al. (2000) Nat. Struct. Biol. (in press)]. The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers. A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family.

SummaryAssay of the adhesion of cultured cells on Toxoplasma gondii tachyzoite protein Western blots identified a major adhesive protein, that migrated at 90 kDa in non-reducing gels. This band comigrated with the previously described microneme protein MIC3. Cellular binding on Western blots was abolished by MIC3-specific monoclonal and polyclonal antibodies. The MIC3 protein affinity purified from tachyzoite lysates bound to the surface of putative host cells. In addition, T. gondii tachyzoites also bound to immobilized MIC3. Immunofluorescence analysis of T. gondii tachyzoite invasion showed that MIC3 was exocytosed and relocalized to the surface of the parasite during invasion. The cDNA encoding MIC3 and the corresponding gene have been cloned, allowing the determination of the complete coding sequence. The MIC3 sequence has been confirmed by affinity purification of the native protein and N-terminal sequencing. The deduced protein sequence contains five partially overlapping EGF-like domains and a chitin binding-like domain, which can be involved in protein±protein or protein± carbohydrate interactions. Taken together, these results suggest that MIC3 is a new microneme adhesin of T. gondii.

The structural gene of the enterococcal peptide antibiotic AS-48 (as-48) has been identified and cloned by using two degenerate 17-mer DNA oligonucleotides on the basis of the amino acid sequences of two peptides obtained by digestion of the antibiotic with Glu-C endoproteinase. That as-48 gene codes for a 105-amino-acid prepeptide, giving rise to a 70-amino-acid mature protein. Comparative analysis demonstrated that the 16-amino-acid sequence of one of the AS48 Glu-C peptides, designated V8-5, was composed of a 12-aminIo-acid sequence corresponding to the C-terminal end sequence (from isoleucine +59 to tryptophan +70 [I+'9 to W+70J) of the prepeptide and terminated in four residues forming the N terminus (M+1 to E+4) of a putative AS-48 propeptide. These data, combined with the characteristics of the gene sequence, strongly suggested that the antibiotic peptide was a 70-residue cyclic molecule. We propose that the AS-48 translated primary product is very likely submitted to a posttranslational modification during secretion (i) by an atypical or a typical signal peptidase that cleaves of a 35-residue or shorter signal peptide, respectively, from the prepeptide molecule and (ii) by the linkage of the methionine residue (M+1) to the C-terminal tryptophan residue (W+70) to obtain the cyclic peptide (a tall-head linkage).For many years, it has been known that enterococci produce inhibitory substances such as bacteriocins and hemolysins that are encoded by large widely disseminated and transmissible plasmids (4,9,13,20,22). These plasmids usually are able to transfer to recipient cells at a relatively high frequency in broth (4,13,24,25). The best-characterized bacteriocin produced by enterococci is the bacteriocin-hemolysin encoded by the sex pheromone plasmid pADi (11,14). The cytolytic system of pADi revealed two protein components, designated A (activator) and L (lytic), able to complement each other extracellularly (11,14) and to contribute to bacterial virulence in an animal model (15).AS-48 is a plasmid-encoded peptide antibiotic produced by Enterococcus faecalis S-48 with broad antimicrobial activities against gram-positive and gram-negative bacteria (6). Some pathogenic bacteria, including Staphylococcus, Enterococcus, and Salmonella species, are highly sensitive to AS-48 (7). Insertion of the peptide into the cytoplasmic membrane of target cells or artificial membranes renders the membrane permeable to small molecules (ions, amino acids), causing the release of cytoplasmic material and the lysis of sensitive cells (8).Because of its strongly cationic properties and biological activity, the AS-48 peptide antibiotic could be related to the group of bacteriocins and peptide antibiotics, mainly produced by lactic acid bacteria, which appear to function by permeabilizing the cytoplasmic membrane. They include lactococcins A, B, and G (12, 27, 39); lactocin S (26); and the group of lantibiotics, e.g., nisin (2, 10), epidermin (34)

Biological research has focused in the past on model organisms and most of the functional genomics studies in the field of plant sciences are still performed on model species or species that are characterized to a great extent. However, numerous non-model plants are essential as food, feed, or energy resource. Some features and processes are unique to these plant species or families and cannot be approached via a model plant. The power of all proteomic and transcriptomic methods, that is, high-throughput identification of candidate gene products, tends to be lost in non-model species due to the lack of genomic information or due to the sequence divergence to a related model organism. Nevertheless, a proteomics approach has a great potential to study non-model species. This work reviews non-model plants from a proteomic angle and provides an outline of the problems encountered when initiating the proteome analysis of a non-model organism. The review tackles problems associated with (i) sample preparation, (ii) the analysis and interpretation of a complex data set, (iii) the protein identification via MS, and (iv) data management and integration. We will illustrate the power of 2DE for non-model plants in combination with multivariate data analysis and MS/MS identification and will evaluate possible alternatives.

Plant aspartic proteinases contain a plant-specific insert (PSI) of about 100 amino acids of unknown function with no similarity with the other aspartic proteinases but with significant similarity with saposins, animal sphingolipid activator proteins. PSI has remained elusive at the protein level, suggesting that it may be removed during processing. To understand the molecular relevance of PSI, the proteolytic processing of cardosin A, the major aspartic proteinase from the flowers of cardoon (Cynara cardunculus L.) was studied. Procardosin A, a 64-kDa cardosin A precursor containing PSI and the prosegment was identified by immunoblotting using monospecific antibodies against PSI and the prosegment. Procardosin A undergoes proteolytic processing as the flower matures. PSI was found to be removed before the prosegment, indicating that during processing the enzyme acquires a structure typical of mammalian or microbial aspartic proteinase proforms. In vitro studies showed that processing of PSI occurs at pH 3.0 and is inhibited by pepstatin A and at pH 7.0. Sequence analysis allowed the identification of the cleavage sites, revealing that PSI is removed entirely, probably by an aspartic proteinase. Cleavage of the PSI scissile bonds requires, however, a conformation specific to the precursor since isolated cardosins and pistil extracts were unable to hydrolyse synthetic peptides corresponding to the cleavage sites. In view of these results, a model for the proteolytic processing of cardosin A is proposed and the molecular and physiological relevance of PSI in plant aspartic proteinase is discussed.Keywords : aspartic protease ; milk-clotting enzyme; cardosin ; proteolytic processing; saposin.Aspartic proteinases (AP) are a widely distributed class of kingdom [5]. In the majority of plants AP are located in seeds, endoproteases that share significant similarities at the amino-whether quiescent or germinating. They are believed to particiacid-sequence level and at the structural level [1, 2]. The typical pate in storage-protein cleavage, which is necessary for germinacharacteristics of the family are an acidic pH optimum, inhibi-tion [5,10]. AP have also been found in leaves of some plants. tion by pepstatin A, preference for bonds between hydrophobic In leaves they have been implicated in mechanisms of defense amino acids, and sequence similarities, in particular the conser-against pathogens [11,12]. Species of the genus Cynara contain vation of the catalytic triads Asp-Thr-Gly or Asp-Ser-Gly [1Ϫ considerable amounts of AP in flowers. Recent data indicate that 4]. AP have been implicated in diverse physiological processes, the major AP from cardoon (Cynara cardunculus L.), cardosin such as digestion or blood-pressure regulation, and in some A, may be involved in the sexual reproduction of the plant [6] pathological conditions, including infection by fungi and retro-and may have a defensive role as well [13]. viruses or cancer [5, 6].A few primary structures deduced from the cDNAs of AP Like many other proteases, A...

The complete primary structure of the peptide antibiotic AS-48 produced by Enterococcus faecalis has heen determined by chemical degradation analysis. The cyclic nature of this 70 residues containing peptide was demonstrated by plasma desorption mass analysis of the generated peptides and electrospray ionisation mass analysis of the native polypeptide. As far as we know, this is the first example of an antibiotic protein cyclised by a tail-head peptide bond formation and not by branching of the polypeptide side chains.