Xylanases hydrolyze the beta-1,4-linked xylose backbone of xylans. They are of increasing interest in the paper and food industries for their pre-bleaching and bio-pulping applications. Such industries demand new xylanases to cover a wider range of cleavage specificity, activity and stability. The catalytic domain of xylanase Xys1 from Streptomyces halstedii JM8 was expressed, purified and crystallized and native data were collected to 1.78 A resolution with an R(merge) of 4.4%. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 34.05, b = 79.60, c = 87.80 A. The structure was solved by the molecular-replacement method using the structure of the homologue Xyl10A from Streptomyces lividans. In a similar manner to other members of its family, Xys1 folds to form a standard (beta/alpha)(8) barrel with the two catalytic functions, the acid/base and the nucleophile, at its C-terminal side. The overall structure is described and compared with those of related xylanases.

Four phospholipases D (PLDs) in the culture supernatants from Streptomyces strains were purified to conduct a comparative study of their thermostabilities. Among the four purified PLDs, the enzyme from Streptomyces halstedii K1 lost its activity at 45 degrees C. PLD from Streptomyces septatus TH-2 was stable at the same temperature. We determined the nucleotide sequence encoding the PLD gene from S. halstedii K1 (K1PLD). The deduced amino acid sequence showed high homology to that of the PLD gene from S. septatus TH-2 (TH-2PLD). By comparison of the optimum temperature and the thermostability among recombinant PLDs, K1PLD, TH-2PLD and T/KPLD that possessed the N-terminus of TH-2PLD and the C-terminus of K1PLD, T/KPLD showed the properties midway between those of K1PLD and TH-2PLD. It was suggested that the 176 amino acids at C-terminus of Streptomyces PLD were important for its thermostability.

Vicenistatin, an antitumor antibiotic isolated from Streptomyces halstedii, is a unique 20-membered macrocyclic lactam with a novel aminosugar vicenisamine. The vicenistatin biosynthetic gene cluster (vin) spanning approximately 64 kbp was cloned and sequenced. The cluster contains putative genes for the aglycon biosynthesis including four modular polyketide synthases (PKSs), glutamate mutase, acyl CoA-ligase, and AMP-ligase. Also found in the cluster are genes of NDP-hexose 4,6-dehydratase and aminotransferase for vicenisamine biosynthesis. For the functional confirmation of the cluster, a putative glycosyltransferase gene product, VinC, was heterologously expressed, and the vicenisamine transfer reaction to the aglycon was chemically proved. A unique feature of the vicenistatin PKS is that the loading module contains only an acyl carrier protein domain, in contrast to other known PKS-loading modules containing certain activation domains. Activation of the starter acyl group by separate polypeptides is postulated as well.

Streptomyces griseus and related species form the biggest but least well-defined clade in the whole Streptomyces 16S rRNA gene tree. Multilocus sequence analysis (MLSA) has shown promising potential for refining Streptomyces systematics. In this investigation, strains of 18 additional S. griseus clade species were analysed and data from a previous pilot study were integrated in a larger MLSA phylogeny. The results demonstrated that MLSA of five housekeeping genes (atpD, gyrB, recA, rpoB and trpB) is better than the previous six-gene scheme, as it provides equally good resolution and stability and is more cost-effective; MLSA using three or four of the genes also shows good resolution and robustness for differentiating most of the strains and is therefore of value for everyday use. MLSA is more suitable for discriminating strains that show >99 % 16S rRNA gene sequence similarity. DNA-DNA hybridization (DDH) between strains with representative MLSA distances revealed a strong correlation between the data of MLSA and DDH. The 70 % DDH value for current species definition corresponds to a five-gene MLSA distance of 0.007, which could be considered as the species cut-off for the S. griseus clade. It is concluded that the MLSA procedure can be a practical, reliable and robust alternative to DDH for the identification and classification of streptomycetes at the species and intraspecies levels. Based on the data from MLSA and DDH, as well as cultural and morphological characteristics, 18 species and three subspecies of the S. griseus clade are considered to be later heterotypic synonyms of 11 genomic species: Streptomyces griseinus and Streptomyces mediolani as synonyms of Streptomyces albovinaceus; Streptomyces praecox as a synonym of Streptomyces anulatus; Streptomyces olivoviridis as a synonym of Streptomyces atroolivaceus; Streptomyces griseobrunneus as a synonym of Streptomyces bacillaris; Streptomyces cavourensis subsp. washingtonensis as a synonym of Streptomyces cyaneofuscatus; Streptomyces acrimycini, Streptomyces baarnensis, Streptomyces caviscabies and Streptomyces flavofuscus as synonyms of Streptomyces fimicarius; Streptomyces flavogriseus as a synonym of Streptomyces flavovirens; Streptomyces erumpens, 'Streptomyces ornatus' and Streptomyces setonii as synonyms of Streptomyces griseus; Streptomyces graminofaciens as a synonym of Streptomyces halstedii; Streptomyces alboviridis, Streptomyces griseus subsp. alpha, Streptomyces griseus subsp. cretosus and Streptomyces luridiscabiei as synonyms of Streptomyces microflavus; and Streptomyces californicus and Streptomyces floridae as synonyms of Streptomyces puniceus.

Acyltransferases (ATs) are key determinants of building block specificity in polyketide biosynthesis. Despite the importance of protein-protein interactions between AT and acyl carrier protein (ACP) during the acyltransfer reaction, the mechanism of ACP recognition by AT is not understood in detail. Herein, we report the crystal structure of AT VinK, which transfers a dipeptide group between two ACPs, VinL and VinP1LdACP, in vicenistatin biosynthesis. The isolated VinK structure showed a unique substrate-binding pocket for the dipeptide group linked to ACP. To gain greater insight into the mechanism of ACP recognition, we attempted to crystallize the VinK-ACP complexes. Because transient enzyme-ACP complexes are difficult to crystallize, we developed a covalent cross-linking strategy using a bifunctional maleimide reagent to trap the VinK-ACP complexes, allowing the determination of the crystal structure of the VinK-VinL complex. In the complex structure, Arg-153, Met-206, and Arg-299 of VinK interact with the negatively charged helix II region of VinL. The VinK-VinL complex structure allows, to our knowledge, the first visualization of the interaction between AT and ACP and provides detailed mechanistic insights into ACP recognition by AT.

Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of �\-amino acid substrates have been obtained for �\-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the �\-amino group of the substrate. In contrast, the �]-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of �]-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp(230) residue is used in the recognition of the �]-amino group of 3-MeAsp similar to �\-amino acid adenylation enzymes. A mutational analysis and structural comparison with �\-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a �]-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a �]-amino acid and provides new mechanistic insights into the selective recognition of �]-amino acids in this family of enzymes.

VinJ is an amidohydrolase belonging to the serine peptidase family that catalyzes the hydrolysis of the terminal aminoacyl moiety of a polyketide intermediate during the biosynthesis of vicenistatin. Herein, we report the crystal structure of VinJ. VinJ possesses a unique hydrophobic tunnel for the recognition of the polyketide chain moiety of its substrate in the cap domain. Taken together with the results of phylogenetic analysis, our results suggest that VinJ represents a new amidohydrolase family that is different from the known �\/�] hydrolase type serine peptidases.

Streptomyces halstedii JM8 Cel2 is an endoglucanase of 28 kDa that is first produced as a protein of 42 kDa (p42) and is later processed at its C-terminus. Cel2 displays optimal activity towards CM-cellulose at pH6 and 50 degrees C and shows no activity against crystalline cellulose or xylan. The N-terminus of p42 shares similarity with cellulases included in family 12 of the beta-glycanases and the C-terminus shares similarity with bacterial cellulose-binding domains included in family II. This latter domain enables the precursor to bind so tightly to Avicel that it can only be eluted by boiling in 10% (w/v) SDS. Another open reading frame (ORF) situated 216 bp downstream from the p42 ORF encodes a protein of 40 kDa (p40) that does not have any clear hydrolytic activity against cellulosic or xylanosic compounds, but shows high affinity for Avicel (crystalline cellulose). The p40 protein is processed in old cultures to give a protein of 35 kDa that does not bind to Avicel. Translation of both ORFs is impaired in Streptomyces coelicolor bldA mutants, suggesting that a TTA codon situated at the fourth position of the first ORF is responsible for this regulation. S1 nuclease protection experiments demonstrate that both ORFs are co-transcribed.

The gene xysA from Streptomyces halstedii JM8 encodes a protein of 461 amino acids (Xys1) which is secreted into the culture supernatant as a protein of 45 kDa (Xys1L). Later, this form is proteolytically processed after residue D-362 to produce the protein Xys1S, which conserves the same xylanolytic activity. The cleavage removes a domain of 99 amino acids that shows similarity to bacterial cellulose binding domains and that allows the protein Xys1L to bind to crystalline cellulose (Avicel). Expression of this monocistronic gene is affected by the carbon source present in the culture medium, xylan being the best inducer. By using an anti-Xys1L serum, we have been able to detect xylanases similar in size to Xys1L and Xys1S in most of the different Streptomyces species analyzed, suggesting the ubiquity of these types of xylanases and their processing mechanism.

The whiE gene cluster of Streptomyces coelicolor, which is related to gene sets encoding the biosynthesis of polycyclic aromatic polyketide antibiotics, determines a spore pigment. Southern blotting using probes from three different parts of the whiE cluster revealed related gene sets in about half of a collection of diverse Streptomyces strains. A 5.2-kb segment of one such cluster, sch, previously shown to determine spore pigmentation in Streptomyces halstedii, was sequenced. Seven open reading frames (ORFs), two of them incomplete, were found. Six of the ORFs resemble the known part of the whiE cluster closely. The derived gene products include a ketosynthase (= condensing enzyme) pair, acyl carrier protein and cyclase, as well as two of unidentified function. The seventh ORF diverges from the main cluster and encodes a protein that resembles a dichlorophenol hydroxylase. Comparison with sequences of related gene sets for the biosynthesis of antibiotics suggests that gene clusters destined to specify pigment production diverged from those destined to specify antibiotics early in the evolution of the Streptomyces genus.

A gene, schC, adjacent to the sch gene cluster encoding the biosynthesis of a polyketide spore pigment in Streptomyces halstedii was sequenced. Its deduced product resembled flavin adenine nucleotide-containing hydroxylases involved in the biosynthesis of polycyclic aromatic polyketide antibiotics and in catabolic pathways of aromatic compounds. When schC was disrupted, the normally green spores of S. halstedii became lilac. An schC-like gene was located in an equivalent position next to a large gene cluster (whiE) known to determine spore pigment in Streptomyces coelicolor A3(2).

Streptomyces halstedii JM8, isolated from straw, produces and secretes into the culture supernatant at least two proteins with hydrolytic activity towards xylan. The cloning of a DNA fragment of this microorganism in several Streptomyces strains permitted us to overproduce both proteins. N-terminal sequence analyses, immunoblot assays, and time course overproduction experiments allowed us to ensure that both xylanases were encoded by the same gene and that the smallest form (35 kDa) originated from the large one (45 kDa) by proteolytic cleavage on the C terminus. The production of both forms was studied in different strains carrying the gene in a multicopy plasmid. The best production was obtained with Streptomyces parvulus transformed with the plasmid pJM9, a pIJ702 derivative, which yielded 144 U/ml. Both forms of the xylanase were purified with a fast-performance liquid chromatography system and characterized biochemically. The optimal pH and temperature, for both, were 6.3 and 60 degrees C, respectively, in 7.5-min assays. Both proteins were highly stable in a wide range of pHs (4 to 10) and temperatures (4 to 50 degrees C); nevertheless, after 1-h incubations, both enzymes lost most of their activity at temperatures over 55 to 60 degrees C. Endoxylanolytic activity was demonstrated in both enzymes, but no beta-xylosidase activity was detected.