Highly efficient synthesis of chiral alcohols with a novel NADH-dependent reductase from Streptomyces coelicolor

An NADH-dependent reductase (ScCR) from Streptomyces coelicolor was discovered by genome mining for carbonyl reductases. ScCR was overexpressed in Escherichia coli BL21, purified to homogeneity and its catalytic properties were studied. This enzyme catalyzed the asymmetric reduction of a broad range of prochiral ketones including aryl ketones, α- and β-ketoesters, with high activity and excellent enantioselectivity (>99% ee) towards β-ketoesters. Among them, ethyl 4-chloro-3-oxobutanoate (COBE) was efficiently converted to ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), an important pharmaceutical intermediate, in water/toluene biphasic system. As much as 600 g/L (3.6 M) of COBE was asymmetrically reduced within 22 h using 2-propanol as a co-substrate for NADH regeneration, resulting in a yield of 93%, an enantioselectivity of >99% ee, and a total turnover number (TTN) of 12,100. These results indicate the potential of ScCR for the industrial production of valuable chiral alcohols.     

Efficient transposition of IS204-derived plasmids in Streptomyces coelicolor

In order to study functional gene expression in Streptomyces coelicolor, a mini-transposon encoding the apramycin resistance gene aac(3)IV within its inverted repeat (IR) boundaries was constructed based on IS204, which was previously identified in the genome of Nocardia asteroides YP21. The mini-transposon and IS204 transposase gene were then put on a kanamycin-resistant conjugative plasmid pDZY101 that can only replicate in Escherichia coli. After mating with S. coelicolor A3(2) M145, resistant colonies arose efficiently on both apramycin and kanamycin plates. Plasmid rescue indicated that entire plasmids were inserted into the M145 genome with cleavage at an inverted repeat junction formed by the right inverted repeat (IRR) and the last 18 bp of the transposase gene, while the left inverted repeat (IRL) was untouched. Southern blot analysis of the mutants using an aac(3)IV gene probe showed that transposition of plasmid pDZY101 was genetically stable, with a single-copy insertion within the S. coelicolor M145 genome. Several mutagenesis libraries of S. coelicolor M145 were constructed using plasmid pDZY101 derivatives and the transposon insertion site was determined. The correlation between novel mutant phenotypes and previously uncharacterized genes was established and these transposon locations were widely scattered around the genome.     

A single-residue mutation destabilizes Vibrio harveyi flavin reductase FRP dimer

Our earlier studies have shown that the Vibrio harveyi flavin reductase FRP undergoes a monomer-dimer equilibrium, and luciferase forms a functional complex with the FRP monomer but not significantly with the dimer. This work is aimed at further investigating the nature and regulation of FRP subunit interactions by computation and site-directed mutagenesis approaches. In silico mutations of a number of residues were performed, and energetic analyses led us to target residue E99, which interacts directly with R113 and R225 from the second subunit of the FRP homodimer, for detailed investigation. E99 was found non-essential to the binding of either the FMN cofactor or the substrates. However, in comparison with the native enzyme, the E99K variant was shown to have an enhanced subunit dissociation as evident from a 44-fold higher K_d for the monomer-dimer equilibrium. The critical role of E99 in the formation of the FRP dimer has thus been demonstrated.     

Organisation of the ribosomal RNA genes in Streptomyces coelicolor A3(2)

Summary Using Southern hybridisation of radiolabelled purified ribosomal RNAs to genomic DNA the ribosomal RNA genes of Streptomyces coelicolor A3(2) were shown to be present in six gene sets. Each gene set contains at least one copy of the 5 S, 16 S and 23 S sequences and in at least two cases these are arranged in the order 16 S-23S-5S. Three gene sets, rrnB, rrnD and rrnF, were isolated by screening a library of S. coelicolor A3(2) DNA. The restriction map of one of these, rrnD, was determined and the nucleotide sequences corresponding to the three rRNAs were localised by Southern hybridisation. The gene order in rrnD is 16S-23S-5S.     

Cloning of a gene encoding flavin reductase coupling with dibenzothiophene monooxygenase through coexpression screening using indigo production as selective indication

The thermophilic dibenzothiophene (DBT)-desulfurizing bacterium, Bacillus subtilis WU-S2B, possesses the ability to convert DBT to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range up to 50 degrees C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and flavin reductase is essential in combination with these flavin-dependent monooxygenases. The recombinant Escherichia coli cells expressing the DBT monooxygenase gene (bdsC) from B. subtilis WU-S2B also oxidize indole to blue pigment indigo in the presence of a heterologous flavin reductase. Thus, to clone a gene encoding flavin reductase from B. subtilis WU-S2B, indigo production by coexpression of the gene with bdsC in E. coli was used as a selection. Using this method, the corresponding gene (frb) was obtained from a recombinant strain forming a blue colony due to indigo production on a nutrient agar plate, and it was confirmed that this gene product Frb exhibited flavin reductase activity. The deduced amino acid sequence of frb consists of 174 amino acid residues and shares 61% identity with that of nitroreductase (YwrO) of Bacillus amyloliquefaciens. In addition, coexpression of frb with the DBT-desulfurization genes (bdsABC) from B. subtilis WU-S2B was critical for high DBT-desulfurizing ability over a wide temperature range of 20-55 degrees C. This coexpression screening using indigo production as selective indication may be widely applicable for cloning novel genes encoding either component of flavin reductase or flavin-dependent monooxygenase which efficiently couples with the other component in two-component monooxygenases.     

Sample preparation for two-dimensional blue native/SDS polyacrylamide gel electrophoresis in the identification of Streptomyces coelicolor cytoplasmic protein complexes

Ammonium sulfate precipitation was tested as a sample preparation step for BN-PAGE analyses of S. coelicolor cytoplasmic protein complexes. A procedure of sample preparation compatible with two-dimensional BN/SDS-PAGE was established and used to visualize protein complexes. To validate the sample preparation procedure, representative protein complexes were identified. Several previously characterized protein complexes were rediscovered and their reported oligomeric states reconfirmed. In addition, we identified new but plausible interactions that have never been reported before. Our work provides useful reference for the wide application of BN-PAGE in protein interaction study.     

The role of Glu196 in the environment around the substrate binding site of leucine aminopeptidase from Streptomyces griseus

To investigate the role of Glu196 of leucine aminopeptidase from Streptomyces griseus (SGAP) in SGAP activation by calcium and substrate specificity, we constructed E196X SGAP by saturation mutagenesis. Most mutations led to the abrogation of SGAP activation by calcium, and substitution with Lys led to a marked increase in activity toward Asp-p-nitroanilide (pNA) and a decrease in that toward Lys-pNA. A similar result was obtained from the investigation using non-calcium-activated enzyme from Streptomyces septatus (SSAP). These results indicate that Glu196 of SGAP is associated with the environment around the substrate binding site besides its role in SGAP activation by calcium.     

Defining the epitope region of a peptide from the Streptomyces coelicolor phosphoenolpyruvate:sugar phosphotransferase system able to bind to the enzyme I

The bacterial PEP:sugar PTS consists of a cascade of several proteins involved in the uptake and phosphorylation of carbohydrates, and in signal transduction pathways. Its uniqueness in bacteria makes the PTS a target for new antibacterial drugs. These drugs can be obtained from peptides or protein fragments able to interfere with the first reaction of the protein cascade: the phosphorylation of the HPr by the first enzyme, the so-called enzyme EI. To that end, we designed a peptide, HPr^9-30, spanning residues 9 to 30 of the intact HPr protein, containing the active site histidine (His-15) and the first α-helix of HPr of Streptomyces coelicolor, HPr^sc. By using fluorescence and circular dichroism, we first determined qualitatively that HPr^sc and HPr^9-30 did bind to EI^sc, the enzyme EI from S. coelicolor. Then, we determined quantitatively the binding affinities of HPr^9-30 and HPr^sc for EI^sc by using ITC and STD-NMR. The STD-NMR experiments indicate that the epitope region of HPr^9-30 was formed by residues Leu-14, His-15, Ile-21, and Val-23. The binding reaction between EI^sc and HPr^sc is enthalpy driven and in other species is entropy driven; further, the affinity of HPr^sc for EI^sc was smaller than in other species. However, the affinity of HPr^9-30 for EI^sc was only moderately lower than that of EI^sc for HPr^sc, suggesting that this peptide could be considered a promising hit compound for designing new inhibitors against the PTS.     

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 °C in Tris–HCl buffer at optimal pH = 7.5 for either ssDNA or dsDNA substrates. It required a divalent cation (Mg²⁺, Co²⁺, Ca²⁺) and its activity was strongly inhibited in the presence of Zn²⁺, Hg²⁺, chelating agents or iodoacetate.     

Substrate specificity of penicillin acylase from Streptomyces lavendulae

The kinetic parameters of several substrates of penicillin acylase from Streptomyces lavendulae have been determined. The enzyme hydrolyses phenoxymethyl penicillin (penicillin V) and other penicillins with aliphatic acyl-chains such as penicillin F, dihydroF, and K. The best substrate was penicillin K (octanoyl penicillin) with a k(cat)/K(m) of 165.3 mM(-1) s(-1). The enzyme hydrolyses also chromogenic substrates as NIPOAB (2-nitro-5-phenoxyacetamido benzoic acid), NIHAB (2-nitro-5-hexanoylamido benzoic acid) or NIOAB (2-nitro-5-octanoylamido benzoic acid), however failed to hydrolyse phenylacetil penicillin (penicillin G) or NIPAB (2-nitro-5-phenylacetamido benzoic acid) and penicillins with polar substituents in the acyl moiety. These results suggest that the structure of the acyl moiety of the substrate is more determinant than the amino moiety for enzyme specificity. The enzyme was inhibited by several organic acids and the extent of inhibition changed with the hydrophobicity of the acid. The best inhibitor was octanoic acid with a K(i) of 0.8 mM. All the results, taking together, point to an active site highly hydrophobic for this penicillin acylase from Streptomyces lavendulae.     

Investigation of the functional properties and regulation of three glutamine synthetase-like genes in Streptomyces coelicolor A3(2)

Streptomyces coelicolor A3(2) has three additional glnA-type genes besides the glutamine synthetase genes glnA (encoding GSI) and glnII (encoding GSII). The aim of this work was to characterize their functional properties and regulation. Sequence analyses revealed that GlnA2, GlnA3, and GlnA4 are dissimilar to S. coelicolor GSI and lack highly conserved amino acid residues involved in catalysis. In heterologous expression experiments, glnA2, glnA3, and glnA4, in contrast to glnA and glnII, were not capable of complementing the l-glutamine auxotrophy of an Escherichia coli glnA mutant. The lack of a conserved sequence motif reflecting adenylylation control of enzyme activity suggests that GlnA2, GlnA3, and GlnA4 are not regulated via adenylyltransferase-mediated modification. In DNA-binding assays, the OmpR-like regulator of nitrogen metabolism GlnRII, which interacts with the glnA and glnII promoters, did not bind to the upstream regions of glnA2, glnA3, and glnA4. These findings support the conclusion that glnA2, glnA3, and glnA4 are not directly involved in l-glutamine synthesis and nitrogen assimilation and are not subject to nitrogen control in S. coelicolor. The glnA3 gene product is similar to FluG, which is required for asexual sporulation in Aspergillus nidulans. However, inactivation of glnA3 does not block morphological differentiation in S. coelicolor.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Characteristics of the surface-located carbohydrate-binding protein CbpC from Streptomyces coelicolor A3(2)

Streptomyces coelicolor A32 produces a 35.6-kDa carbohydrate-binding protein (named CbpC) in the presence of cellobiose, cellulose or chitin as sole carbon source. The protein was found secreted (a typical signal sequence was present at the N-terminus) and linked to the peptidoglycan layer of the mycelia. At its C-terminal end a putative cell-wall sorting signal was identified, consisting of (1) Streptomyces specific recognition site for a transpeptidase (LAETG instead of generic LPXTP consensus), (2) a hydrophobic region and (3) a tail of positively charged residues. The deletion of this sorting signal abolished the cell-wall attachment because the resulting CbpC-form was found extracellular. After purification this protein was shown to interact strongly with crystalline cellulose; different crystalline chitin-forms were recognised moderately and chitosan not. As demonstrated by analysing further truncated CbpC-forms a glycine-aspartate/serine rich region, which separates the carbohydrate-binding module from the sorting signal, plays an important role in protein stability.     

A cold-active esterase of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2): from genome sequence to enzyme activity

The genome sequence of Streptomyces coelicolor A3(2) contains 51 putative lipase and esterase genes mostly of unknown function. The gene estB (locus SCO 6966) was expressed as a His-tagged protein in E. coli. Esterase B was active at low temperatures exerting its maximum activity at 30°C and retaining more than 25% of its activity at 4°C. The optimum pH was 8–8.5. The enzyme was active against short synthetic p-nitrophenylesters (C₂–C₁₀) with maximum activity towards the acetate ester (C₂). The esterase was tested on 13 series of racemic esters of potential interest for the synthesis of chiral pharmaceutical compounds. 4 of the series were substrates and a modest degree of enantioselectivity was observed (enantiomeric ratios of 1.1–1.9).     

The SGNH-hydrolase of Streptomyces coelicolor has (aryl)esterase and a true lipase activity

The Streptomyces coelicolor A3(2) gene SCI11.14c was overexpressed and purified as a His-tagged protein from heterologous host, Streptomyces lividans. The purification procedure resulted in 34.1-fold increase in specific activity with an overall yield of 21.4%. Biochemical and physical properties of the purified enzyme were investigated and it was shown that it possesses (aryl)esterase and a true lipase activity. The enzyme was able to hydrolyze p-nitrophenyl-, α- and β-naphthyl esters and poly(oxyethylene) sorbitan monoesters (Tween 20–80). It showed pronounced activity towards p-nitrophenyl and α- and β-naphthyl esters of C₁₂–C₁₆. Higher activity was observed with α-naphthyl esters. The enzyme hydrolyzed triolein (specific activity: 91.9 U/mg) and a wide range of oils with a preference for those having higher content of linoleic or oleic acid (C18:2; C18:1, cis). The active-site serine specific inhibitor 3,4-dichloroisocoumarin (DCI) strongly inhibited the enzyme, while tetrahydrofurane and 1,4-dioxane significantly increased (2- and 4- fold, respectively) hydrolytic activity of lipase towards p-nitrophenyl caprylate. The enzyme exhibited relatively high temperature optimum (55 °C) and thermal stability. CD analysis revealed predominance of α-helical structure (54% α-helix, 21% β-sheet) and a T_m value at 66 °C.     

Kinetic characterization of an oxidative,cooperative HMG-CoA reductase from Burkholderia cenocepacia

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a key enzyme in endogenous cholesterol biosynthesis in mammals and isoprenoid biosynthesis via the mevalonate pathway in other eukaryotes, archaea and some eubacteria. In most organisms that express this enzyme, it catalyzes the NAD(P)H-dependent reduction of HMG-CoA to mevalonate. We have cloned and characterized the 6x-His-tagged HMGR from the opportunistic lung pathogen Burkholderia cenocepacia. Kinetic characterization shows that the enzyme prefers NAD(H) over NADP(H) as a cofactor, suggesting an oxidative physiological role for the enzyme. This hypothesis is supported by the fact that the Burkholderia cenocepacia genome lacks the genes for the downstream enzymes of the mevalonate pathway. The enzyme exhibits positive cooperativity toward the substrates of the reductive reaction, but the oxidative reaction exhibits unusual double-saturation kinetics, distinctive among characterized HMG-CoA reductases. The unusual kinetics may arise from the presence of multiple active oligomeric states, each with different V_max values.     

Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase

Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3 ^– null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.     

Structural and functional characterization of the Geobacillus copper nitrite reductase: Involvement of the unique N-terminal region in the interprotein electron transfer with its redox partner

The crystal structures of copper-containing nitrite reductase (CuNiR) from the thermophilic Gram-positive bacterium Geobacillus kaustophilus HTA426 and the amino (N)-terminal 68 residue-deleted mutant were determined at resolutions of 1.3 Å and 1.8 Å, respectively. Both structures show a striking resemblance with the overall structure of the well-known CuNiRs composed of two Greek key β-barrel domains; however, a remarkable structural difference was found in the N-terminal region. The unique region has one β-strand and one α-helix extended to the northern surface of the type-1 copper site. The superposition of the Geobacillus CuNiR model on the electron-transfer complex structure of CuNiR with the redox partner cytochrome c₅₅₁ in other denitrifier system led us to infer that this region contributes to the transient binding with the partner protein during the interprotein electron transfer reaction in the Geobacillus system. Furthermore, electron-transfer kinetics experiments using N-terminal residue-deleted mutant and the redox partner, Geobacillus cytochrome c₅₅₁, were carried out. These structural and kinetics studies demonstrate that the region is directly involved in the specific partner recognition.