Biophysical characterization of the enzyme I of the Streptomyces coelicolor phosphoenolpyruvate:sugar phosphotransferase system

The first protein in the bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system is the homodimeric 60-kDa enzyme I (EI), which autophosphorylates in the presence of PEP and Mg2+. The conformational stability and structure of the EI from Streptomyces coelicolor, EI(sc), were explored in the absence and in the presence of its effectors by using several biophysical probes (namely, fluorescence, far-ultraviolet circular dichroism, Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry) and computational approaches. The structure of EI(sc) was obtained by homology modeling of the isolated N- and C-terminal domains of other EI proteins. The experimental results indicate that at physiological pH, the dimeric EI(sc) had a well-folded structure; however, at low pH, EI(sc) showed a partially unfolded state with the features of a molten globule, as suggested by fluorescence, far-ultraviolet circular dichroism, FTIR, and 8-anilino-1-naphthalene-sulfonic acid binding. The thermal stability of EI(sc), in the absence of PEP and Mg2+, was maximal at pH 7. The presence of PEP and Mg2+ did not change substantially the secondary structure of the protein, as indicated by FTIR measurements. However, quenching experiments and proteolysis patterns suggest conformational changes in the presence of PEP; furthermore, the thermal stability of EI(sc) was modified depending on the effector added. Our approach suggests that thermodynamical analysis might reveal subtle conformational changes.     

Intracellular protease activity during the growth of Streptomyces coelicolor

Multiple intracellular proteases were produced by Streptomyces coelicolor throughout growth as surface cultures. Zymography revealed two constitutive, gelatinolytic proteases of approximate molecular masses 32.5 and 36.5 kDa. In addition, transient expression of a large (183.5 kDa) protease preceded aerial mycelium formation and following this, during sporulation, an additional protease of mass 27.5 kDa was produced.     

Functional characterization of Streptomyces coelicolor FtsY

This study indicated that the N-terminus was dispensable for FtsY GTPase activity, and that the N-domain plays an essential role in the GTPase activity of the NG domain. In addition, the S.scoelicolor FtsY was able to restore function in an E. coli mutant. However, its NG domain was unable to play any roles.     

Adenosine deaminase from Streptomyces coelicolor: recombinant expression, purification and characterization

The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr=48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s?1 at 30 °C. Since adenine is deaminated ～103 slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-β-D-arabinofuranosyl-adenine (ara-A), 5'-AMP, 5'-ADP and 5'-ATP, are not substrates for the enzyme. Coformycin and 2'-deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common α/β barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism.     

An exodeoxyribonuclease from Streptomyces coelicolor: expression, purification and biochemical characterization

Streptomyces coelicolor A3(2) produces several intra and extracellular enzymes with deoxyribonuclease activities. The examined N-terminal amino acid sequence of one of extracellular DNAases (TVTSVNVNGLL) and database search on S. coelicolor genome showed a significant homology to the putative secreted exodeoxyribonuclease. The corresponding gene (exoSc) was amplified, cloned, expressed in Escherichia coli, purified to homogeneity and characterized. Exonuclease recExoSc degraded chromosomal, linear dsDNA with 3'-overhang ends, linear ssDNA and did not digest linear dsDNA with blunt ends, supercoiled plasmid ds nor ssDNA. The substrate specificity of recExoSc was in the order of dsDNA>ssDNA>3'-dAMP. The purified recExoSc was not a metalloprotein and exhibited neither phosphodiesterase nor RNase activity. It acted as 3'-phosphomonoesterase only at 3'-dAMP as a substrate. The optimal temperature for its activity was 57 degrees C in Tris-HCl buffer at optimal pH=7.5 for either ssDNA or dsDNA substrates. It required a divalent cation (Mg(2+), Co(2+), Ca(2+)) and its activity was strongly inhibited in the presence of Zn(2+), Hg(2+), chelating agents or iodoacetate.     

The purification and characterization of 3-dehydroquinase from Streptomyces coelicolor.

The enzyme 3-dehydroquinase was purified over 4000-fold to homogeneity from Streptomyces coelicolor. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of SDS was 16,000. The native Mr estimated by gel filtration on a Superose 6 column was 209,000, indicating that the enzyme is a large oligomer. The enzyme was found to be extremely thermostable. This stability, along with the structural and kinetic properties of the enzyme, suggest that it is very similar to the quinate-inducible 3-dehydroquinase found in Neurospora crassa and Aspergillus nidulans. This similarity was confirmed by direct N-terminal sequencing.     

Oxygen-reducing enzyme cathodes produced from SLAC, a small laccase from Streptomyces coelicolor

The bacterially-expressed laccase, small laccase (SLAC) of Streptomyces coelicolor, was incorporated into electrodes of both direct electron transfer (DET) and mediated electron transfer (MET) designs for application in biofuel cells. Using the DET design, enzyme redox kinetics were directly observable using cyclic voltammetry, and a redox potential of 0.43 V (SHE) was observed. When mediated by an osmium redox polymer, the oxygen-reducing cathode retained maximum activity at pH 7, producing 1.5 mA/cm2 in a planar configuration at 900 rpm and 40 degrees C, thus outperforming enzyme electrodes produced using laccase from fungal Trametes versicolor (0.2 mA/cm2) under similar conditions. This improvement is directly attributable to differences in the kinetics of SLAC and fungal laccases. Maximum stability of the mediated SLAC electrode was observed at pH above the enzyme's relatively high isoelectric point, where the anionic enzyme molecules could form an electrostatic adduct with the cationic mediator. Porous composite SLAC electrodes with increased surface area produced a current density of 6.25 mA/cm2 at 0.3 V (SHE) under the above conditions.     

Recombinant xylanase from Streptomyces coelicolor Ac-738: characterization and the effect on xylan-containing products

A xylanase gene was isolated from the genomic DNA of Streptomyces coelicolor Ac-738. The 723-bp full-length gene encoded a 241-amino acid peptide consisting of a 49-residue putative TAT signal peptide and a glycoside hydrolase family-11 domain. The mature enzyme called XSC738 was expressed in Escherichia coli M15[pREP4]. The electrophoretically homogeneous protein with a specific activity of 167 U/mg for beechwood xylan was purified. The pH optimum of XSC738 was at pH 6; a high activity was retained within a pH range of 4.5–8.5. The enzyme was thermostable at 50–60 °C and retained an activity at pH 3.0–7.0. Xylanase XSC738 was activated by Mn²⁺, Co²⁺ and largely inhibited by Cd²⁺, SDS and EDTA. The products of xylan hydrolysis were mainly xylobiose, xylotriose, xylopentaose and xylohexose. Xylotetraose appeared as a minor product. Processing of such agricultural xylan-containing products as wheat, oats, soy flour and wheat bran by xylanase resulted in an increased content of sugars.     

Functional characterization and metal ion specificity of the metal-citrate complex transporter from Streptomyces coelicolor

Secondary transporters of citrate in complex with metal ions belong to the bacterial CitMHS family, about which little is known. The transport of metal-citrate complexes in Streptomyces coelicolor has been investigated. The best cofactor for citrate uptake in Streptomyces coelicolor is Fe(3+), but uptake was also noted for Ca(2+), Pb(2+), Ba(2+), and Mn(2+). Uptake was not observed with the Mg(2+), Ni(2+), or Co(2+) cofactor. The transportation of iron- and calcium-citrate makes these systems unique among the CitMHS family members reported to date. No complementary uptake akin to that observed for the CitH (Ca(2+), Ba(2+), Sr(2+)) and CitM (Mg(2+), Ni(2+), Mn(2+), Co(2+), Zn(2+)) systems of Bacillus subtilis was noted. Competitive experiments using EGTA confirmed that metal-citrate complex formation promoted citrate uptake. Uptake of free citrate was not observed. The open reading frame postulated as being responsible for the metal-citrate transport observed in Streptomyces coelicolor was cloned and overexpressed in Escherichia coli strains with the primary Fe(3+)-citrate transport system (fecABCDE) removed. Functional expression was successful, with uptake of Ca(2+)-citrate, Fe(3+)-citrate, and Pb(2+)-citrate observed. No free-citrate transport was observed in IPTG (isopropyl-beta-d-thiogalactopyranoside)-induced or -uninduced E. coli. Metabolism of the Fe(3+)-citrate and Ca(2+)-citrate complexes, but not the Pb(2+)-citrate complex, was observed. Rationalization is based on the difference in metal-complex coordination upon binding of the metal by citrate.     

Biosynthesis of the earthy odorant geosmin by a bifunctional Streptomyces coelicolor enzyme

Geosmin is responsible for the characteristic odor of moist soil, as well as off-flavors in drinking water and foodstuffs. Geosmin is generated from farnesyl diphosphate (FPP, 2) by an enzyme that is encoded by the SCO6073 gene in the soil organism Streptomyces coelicolor A32 (ref. 3). We have now shown that the recombinant N-terminal half of this protein catalyzes the Mg2+-dependent cyclization of FPP to germacradienol and germacrene D, while the highly homologous C-terminal domain, previously thought to be catalytically silent, catalyzes the Mg2+-dependent conversion of germacradienol to geosmin. Site-directed mutagenesis confirmed that the N- and C-terminal domains each harbor a distinct, independently functioning active site. A mutation in the N-terminal domain of germacradienol-geosmin synthase of a catalytically essential serine to alanine results in the conversion of FPP to a mixture of sesquiterpenes that includes an aberrant product identified as isolepidozene, which was previously suggested to be an enzyme-bound intermediate in the cyclization of FPP to germacradienol.     

Isolation and partial characterization of SSI-like protease inhibitors from Streptomyces

We attempted to screen a series of Streptomyces subtilisin inhibitor-like (SIL) proteins among several Streptomyces strains by using a highly sensitive assay system established by us. Of six randomly tested strains, four were found to produce SIL inhibitors as their major secreted proteins, suggesting that they might be distributed in a high frequency among this genus. Three inhibitors exhibited inhibition of both subtilisin BPN' and trypsin. Comparison of the amino terminal sequences of these isolated proteins with those of other reported SIL inhibitors revealed that the beta 1- and beta 2-sheets in SSI were highly conserved.     

The flavin reductase ActVB from Streptomyces coelicolor: characterization of the electron transferase activity of the flavoprotein form

The flavin reductase ActVB is involved in the last step of actinorhodin biosynthesis in Streptomyces coelicolor. Although ActVB can be isolated with some FMN bound, this form was not involved in the flavin reductase activity. By studying the ferric reductase activity of ActVB, we show that its FMN-bound form exhibits a proper enzymatic activity of reduction of iron complexes by NADH. This shows that ActVB active site exhibits a dual property with regard to the FMN. It can use it as a substrate that goes in and off the active site or as a cofactor to provide an electron transferase activity to the polypeptide.     

Identification of a monooxygenase from Streptomyces coelicolor A3(2) involved in biosynthesis of actinorhodin: purification and characterization of the recombinant enzyme.

The oxidation of phenols to quinones is an important reaction in the oxidative tailoring of many aromatic polyketides from bacterial and fungal systems. Sequence similarity between ActVA-Orf6 protein from the actinorhodin biosynthetic cluster and the previously characterized TcmH protein that is involved in tetracenomycin biosynthesis suggested that ActVA-Orf6 might catalyze this transformation as a step in actinorhodin biosynthesis. To investigate the role of ActVA-Orf6 in this oxidation, we have expressed the actVA-Orf6 gene in Escherichia coli and purified and characterized the recombinant protein. ActVA-Orf6 was shown to catalyze the monooxygenation of the tetracenomycin intermediate TcmF1 to TcmD3, strongly suggesting that it catalyzes oxidation of a similar intermediate in actinorhodin biosynthesis. The monooxygenase obeys simple reaction kinetics and has a Km of 4.8 +/- 0.9 microM, close to the figure reported for the homologous enzyme TcmH. The enzyme contains no prosthetic groups and requires only molecular oxygen to catalyze the oxidation. Site-directed mutagenesis was used to investigate the role of histidine residues thought to be important in the reaction; mutants lacking His-52 displayed much-reduced activity, consistent with the proposed mechanistic hypothesis that this histidine acts as a general base during catalysis.     

Phosphoglycerate mutase from Streptomyces coelicolor A3(2): purification and characterization of the enzyme and cloning and sequence analysis of the gene.

The enzyme 3-phosphoglycerate mutase was purified 192-fold from Streptomyces coelicolor, and its N-terminal sequence was determined. The enzyme is tetrameric with a subunit Mr of 29,000. It is 2,3-bisphosphoglycerate dependent and inhibited by vanadate. The gene encoding the enzyme was cloned by using a synthetic oligonucleotide probe designed from the N-terminal peptide sequence, and the complete coding sequence was determined. The deduced amino acid sequence is 64% identical to that of the phosphoglycerate mutase of Saccharomyces cerevisiae and has substantial identity to those of other phosphoglycerate mutases.     

Discovery, characterization, and kinetic analysis of an alditol oxidase from Streptomyces coelicolor

A gene encoding an alditol oxidase was found in the genome of Streptomyces coelicolor A3(2). This newly identified oxidase, AldO, was expressed at extremely high levels in Escherichia coli when fused to maltose-binding protein. AldO is a soluble monomeric flavoprotein with subunits of 45.1 kDa, each containing a covalently bound FAD cofactor. From sequence alignments with other flavoprotein oxidases, it was found that AldO contains a conserved histidine (His(46)) that is typically involved in covalent FAD attachment. Covalent FAD binding is not observed in the H46A AldO mutant, confirming its role in covalent attachment of the flavin cofactor. Steady-state kinetic analyses revealed that wild-type AldO is active with several polyols. The alditols xylitol (K(m) = 0.32 mm, k(cat) = 13 s(-1)) and sorbitol (K(m) = 1.4 mm, k(cat) = 17 s(-1)) are the preferred substrates. From pre-steady-state kinetic analyses, using xylitol as substrate, it can be concluded that AldO mainly follows a ternary complex kinetic mechanism. Reduction of the flavin cofactor by xylitol occurs at a relatively high rate (99 s(-1)), after which a second kinetic event is observed, which is proposed to represent ring closure of the formed aldehyde product, yielding the hemiacetal of d-xylose. Reduced AldO readily reacts with molecular oxygen (1.7 x 10(5) m(-1) s(-1)), which confirms that the enzyme represents a true flavoprotein oxidase.     

Physical characterization of SCP1, a giant linear plasmid from Streptomyces coelicolor.

SCP1, coding for the methylenomycin biosynthesis genes in Streptomyces coelicolor, was shown to be a giant linear plasmid of 350 kb with a copy number of about four by analysis with pulsed-field gel electrophoresis. A detailed physical map of SCP1 was constructed by extensive digestion with six restriction endonucleases, by DNA hybridization experiments, and finally by cloning experiments. SCP1 has unusually long terminal inverted repeats of 80 kb on both ends and an insertion sequence at the end of the right terminal inverted repeat. Analysis by pulsed-field gel electrophoresis in agarose containing sodium dodecyl sulfate revealed that a protein is bound to the terminal 4.1-kb SpeI fragments derived from both ends of SCP1. Treatment with lambda exonuclease or exonuclease III and SpeI digestion also indicated that the 5' ends of SCP1 are attached to a protein.