Characterization of a novel carboxylesterase from Streptomyces lividans TK24 and site-directed mutagenesis for its thermostability.

As an industrial enzyme that catalyzes the formation and cleavage of ester bonds, carboxylesterase has attracted attention in fine chemistry, pharmaceutical, biological energy and bioremediation fields. However, the weak thermostability limits their further developments in industrial applications. In this work, a novel carboxylesterase (EstF) from Streptomyces lividans TK24, belonging to family XVII, was acquired by successfully heterologous expressed and biochemically identified. The EstF exhibited optimal activity at 55 °C, pH 9.0 and excellent catalytic performances (Km = 0.263 mM, kcat/Km = 562.3 s-1 mM-1 for p-nitrophenyl acetate (pNPA2) hydrolysis). Besides, the EstF presented exceptionally high thermostability with a half-life of 387.23 h at 55 °C and 2.86 h at 100 °C. Furthermore, the EstF was modified to obtain EstFP144G using the site-directed mutation technique to investigate the effect of single glycine on thermostability. Remarkably, the mutant EstFP144G displayed a 5.10-fold increase of half-life at 100 °C versus wild-type without affecting catalytic performance. Structural analysis implied that the glycine introduction could release a steric strain and induce cooperative effects between distal residues to increase the thermostability. Therefore, the thermostable EstF and EstFP144G with prominently catalytic characteristics have potential industrial applications and the introduction of a single glycine strategy opens up alternative avenues for the thermostability engineering of other enzymes.

The oligomeric states of dye-decolorizing peroxidases from Streptomyces lividans and their implications for mechanism of substrate oxidation.

A common evolutionary mechanism in biology to drive function is protein oligomerization. In prokaryotes, the symmetrical assembly of repeating protein units to form homomers is widespread, yet consideration in vitro of whether such assemblies have functional or mechanistic consequences is often overlooked. Dye-decolorizing peroxidases (DyPs) are one such example, where their dimeric α + ß barrel units can form various oligomeric states, but the oligomer influence, if any, on mechanism and function has received little attention. In this work, we have explored the oligomeric state of three DyPs found in Streptomyces lividans, each with very different mechanistic behaviors in their reactions with hydrogen peroxide and organic substrates. Using analytical ultracentrifugation, we reveal that except for one of the A-type DyPs where only a single sedimenting species is detected, oligomer states ranging from homodimers to dodecamers are prevalent in solution. Using cryo-EM on preparations of the B-type DyP, we determined a 3.02 Å resolution structure of a hexamer assembly that corresponds to the dominant oligomeric state in solution as determined by analytical ultracentrifugation. Furthermore, cryo-EM data detected sub-populations of higher-order oligomers, with one of these formed by an arrangement of two B-type DyP hexamers to give a dodecamer assembly. Our solution and structural insights of these oligomer states provide a new framework to consider previous mechanistic studies of these DyP members and are discussed in terms of long-range electron transfer for substrate oxidation and in the "storage" of oxidizable equivalents on the heme until a two-electron donor is available.

Characterization of Amycolatopsis 75iv2 dye-decolorizing peroxidase on O-glycosides.

Dye-decolorizing peroxidases are heme peroxidases with a broad range of substrate specificity. Their physiological function is still largely unknown, but a role in the depolymerization of plant cell wall polymers has been widely proposed. Here, a new expression system for bacterial dye-decolorizing peroxidases as well as the activity with previously unexplored plant molecules are reported. The dye-decolorizing peroxidase from Amycolatopsis 75iv2 (DyP2) was heterologously produced in the Gram-positive bacterium Streptomyces lividans TK24 in both intracellular and extracellular forms without external heme supplementation. The enzyme was tested on a series of O-glycosides, which are plant secondary metabolites with a phenyl glycosidic linkage. O-glycosides are of great interest, both for studying the compounds themselves and as potential models for studying specific lignin-carbohydrate complexes. The primary DyP reaction products of salicin, arbutin, fraxin, naringin, rutin, and gossypin were oxidatively coupled oligomers. A cleavage of the glycone moiety upon radical polymerization was observed when using arbutin, fraxin, rutin, and gossypin as substrates. The amount of released glucose from arbutin and fraxin reached 23% and 3% of the total substrate, respectively. The proposed mechanism suggests a destabilization of the ether linkage due to the localization of the radical in the para position. In addition, DyP2 was tested on complex lignocellulosic materials such as wheat straw, spruce, willow, and purified water-soluble lignin fractions, but no remarkable changes in the carbohydrate profile were observed, despite obvious oxidative activity. The exact action of DyP2 on such lignin-carbohydrate complexes therefore remains elusive. IMPORTANCE: Peroxidases require correct incorporation of the heme cofactor for activity. Heterologous overproduction of peroxidases often results in an inactive enzyme due to insufficient heme synthesis by the host organism. Therefore, peroxidases are incubated with excess heme during or after purification to reconstitute activity. S. lividans as a production host can produce fully active peroxidases both intracellularly and extracellularly without the need for heme supplementation. This reduces the number of downstream processing steps and is beneficial for more sustainable production of industrially relevant enzymes. Moreover, this research has extended the scope of dye-decolorizing peroxidase applications by studying naturally relevant plant secondary metabolites and analyzing the formed products. A previously overlooked artifact of radical polymerization leading to the release of the glycosyl moiety was revealed, shedding light on the mechanism of DyP peroxidases. The key aspect is the continuous addition, rather than the more common approach of a single addition, of the cosubstrate, hydrogen peroxide. This continuous addition allows the peroxidase to complete a high number of turnovers without self-oxidation.

Antisense RNA Interference-Enhanced CRISPR/Cas9 Base Editing Method for Improving Base Editing Efficiency in Streptomyces lividans 66.

CRISPR/Cas9-mediated base editors, based on cytidine deaminase or adenosine deaminase, are emerging genetic technologies that facilitate genomic manipulation in many organisms. Since base editing is free from DNA double-strand breaks (DSBs), it has certain advantages, such as a lower toxicity, compared to the traditional DSB-based genome engineering technologies. In terms of Streptomyces, a base editing method has been successfully applied in several model and non-model species, such as Streptomyces coelicolor and Streptomyces griseofuscus. In this study, we first proved that BE2 (rAPOBEC1-dCas9-UGI) and BE3 (rAPOBEC1-nCas9-UGI) were functional base editing tools in Streptomyces lividans 66, albeit with a much lower editing efficiency compared to that of S. coelicolor. Uracil generated in deamination is a key intermediate in the base editing process, and it can be hydrolyzed by uracil DNA glycosidase (UDG) involved in the intracellular base excision repair, resulting in a low base editing efficiency. By knocking out two endogenous UDGs (UDG1 and UDG2), we managed to improve the base editing efficiency by 3.4-67.4-fold among different loci. However, the inactivation of UDG is detrimental to the genome stability and future application of engineered strains. Therefore, we finally developed antisense RNA interference-enhanced CRISPR/Cas9 Base Editing method (asRNA-BE) to transiently disrupt the expression of uracil DNA glycosidases during base editing, leading to a 2.8-65.8-fold enhanced editing efficiency and better genome stability. Our results demonstrate that asRNA-BE is a much better editing tool for base editing in S. lividans 66 and might be beneficial for improving the base editing efficiency and genome stability in other Streptomyces strains.

Serial Femtosecond Zero Dose Crystallography Captures a Water-Free Distal Heme Site in a Dye-Decolorising Peroxidase to Reveal a Catalytic Role for an Arginine in FeIV =O Formation.

Obtaining structures of intact redox states of metal centers derived from zero dose X-ray crystallography can advance our mechanistic understanding of metalloenzymes. In dye-decolorising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues aspartate and arginine in the heterolysis of peroxide to form the catalytic intermediate compound I (FeIV =O and a porphyrin cation radical). Using serial femtosecond X-ray crystallography (SFX), we have determined the pristine structures of the FeIII and FeIV =O redox states of a B-type DyP. These structures reveal a water-free distal heme site that, together with the presence of an asparagine, imply the use of the distal arginine as a catalytic base. A combination of mutagenesis and kinetic studies corroborate such a role. Our SFX approach thus provides unique insight into how the distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxide heterolysis.

Artificial iron hydrogenase made by covalent grafting of Knölker's complex into xylanase: Application in asymmetric hydrogenation of an aryl ketone in water.

We report a new artificial hydrogenase made by covalent anchoring of the iron Knölker's complex to a xylanase S212C variant. This artificial metalloenzyme was found to be able to catalyze efficiently the transfer hydrogenation of the benchmark substrate trifluoroacetophenone by sodium formate in water, yielding the corresponding secondary alcohol as a racemic. The reaction proceeded more than threefold faster with the XlnS212CK biohybrid than with the Knölker's complex alone. In addition, efficient conversion of trifluoroacetophenone to its corresponding alcohol was reached within 60 H with XlnS212CK, whereas a ≈2.5-fold lower conversion was observed with Knölker's complex alone as catalyst. Moreover, the data were rationalized with a computational strategy suggesting the key factors of the selectivity. These results suggested that the Knölker's complex was most likely flexible and could experience free rotational reorientation within the active-site pocket of Xln A, allowing it to access the subsite pocket populated by trifluoroacetophenone.

A subtle structural change in the distal haem pocket has a remarkable effect on tuning hydrogen peroxide reactivity in dye decolourising peroxidases from Streptomyces lividans.

Dye decolourising peroxidases (DyPs) are oxidative haem containing enzymes that can oxidise organic substrates by first reacting with hydrogen peroxide. Herein, we have focused on two DyP homologs, DtpAa and DtpA, from the soil-dwelling bacterium Streptomyces lividans. By using X-ray crystallography, stopped-flow kinetics, deuterium kinetic isotope studies and EPR spectroscopy, we show that both DyPs react with peroxide to form compound I (a FeIV[double bond, length as m-dash]O species and a porphyrin π-cation radical), via a common mechanism, but the reactivity and rate limits that define the mechanism are markedly different between the two homologs (DtpA forms compound I rapidly, no kinetic isotope effect; DtpAa 100-fold slower compound I formation and a distinct kinetic isotope effect). By determining the validated ferric X-ray structure of DtpAa and comparing it with the ferric DtpA structure, we attribute the kinetic differences to a subtle structural repositioning of the distal haem pocket Asp side chain. Through site-directed mutagenesis we show the acid-base catalyst responsible for proton-transfer to form compound I comprises a combination of a water molecule and the distal Asp. Compound I formation in the wild-type enzymes as well as their distal Asp variants is pH dependent, sharing a common ionisation equilibrium with an apparent pKa of ∼4.5-5.0. We attribute this pKa to the deprotonation/protonation of the haem bound H2O2. Our studies therefore reveal a mechanism for compound I formation in which the rate limit may be shifted from peroxide binding to proton-transfer controlled by the distal Asp position and the associated hydrogen-bonded water molecules.

New AMP-forming acid:CoA ligases from Streptomyces lividans, some of which are posttranslationally regulated by reversible lysine acetylation.

In nature, organic acids are a commonly used source of carbon and energy. Many bacteria use AMP-forming acid:CoA ligases to convert organic acids into their corresponding acyl-CoA derivatives, which can then enter metabolism. The soil environment contains a broad diversity of organic acids, so it is not surprising that bacteria such as Streptomyces lividans can activate many of the available organic acids. Our group has shown that the activity of many acid:CoA ligases is posttranslationally controlled by acylation of an active-site lysine. In some cases, the modification is reversed by deacylases of different types. We identified eight new acid:CoA ligases in S. lividans TK24. Here, we report the range of organic acids that each of these enzymes can activate, and determined that two of the newly identified CoA ligases were under NAD+ -dependent sirtuin deacylase reversible lysine (de)acetylation control, four were not acetylated by two acetyltransferases used in this work, and two were acetylated but not deacetylated by sirtuin. This work provides insights into the broad organic-acid metabolic capabilities of S. lividans, and sheds light into the control of the activities of CoA ligases involved in the activation of organic acids in this bacterium.

Functional identification of a Streptomyces lividans FKBP-like protein involved in the folding of overproduced secreted proteins.

Some bacterial peptidyl-prolyl cis/trans isomerases (PPIases) are involved in secretory protein folding after the translocation step. Streptomyces lividans has been used as a host for engineering extracellular overproduction of homologous and heterologous proteins in industrial applications. Although the mechanisms governing the major secretory pathway (Sec route) and the minor secretory pathway (Tat route) are reasonably well described, the function of proteins responsible for the extracellular secretory protein folding is not characterized as yet. We have characterized a Tat-dependent S. lividans FK506-binding protein-like lipoprotein (FKBP) that has PPIase activity. A mutant in the sli-fkbp gene induces a secretion stress response and affects secretion and activity of the Sec-dependent protein α-amylase. Additionally, propagation in high copy number of the sli-fkbp gene has a positive effect on the activity of both the overproduced α-amylase and the overproduced Tat-dependent agarase, both containing proline cis isomers. Targeted proteomic analyses showed that a relevant group of secreted proteins in S. lividans TK21 are affected by Sli-FKBP, revealing a wide substrate range. The results obtained indicate that, regardless of the secretory route used by proteins in S. lividans, adjusting the expression of sli-fkbp may facilitate folding of dependent proteins when engineering Streptomyces strains for the overproduction of homologous or heterologous secretory proteins.

Rational Design of Hybrid Natural Products by Utilizing the Promiscuity of an Amide Synthetase.

WS9326A and annimycin are produced by Streptomyces asterosporus DSM 41452. WS9326A is a nonribosomal peptide synthetase-(NRPS-) derived depsipeptide containing a cinnamoyl moiety, while annimycin is a linear polyketide bearing a 2-amino-3-hydroxycyclopent-2-enone (C5N) group. Both gene clusters have been sequenced and annotated. In this study, we show that the amide synthetase Ann1, responsible for attaching the C5N unit during annimycin biosynthesis, has the ability to catalyze fortuitous side reactions to polyenoic acids in addition to its main reaction. Novel compounds were generated by feeding experiments and in vitro studies. We also rationally designed a hybrid natural product consisting of the cinnamoyl moiety of WS9326A and the C5N moiety of annimycin by creating a mutant of S. asterosporus that retains genes encoding biosynthesis of the C5N unit of annimycin and the cinnamoyl group of WS9326A. The promiscuity of Ann1 also proved useful for trapping compounds that arise from acyl-ACP intermediates, which occur in the biosynthesis of the cinnamoyl moiety of WS9326A, by hydrolysis. In this pathway, we postulate that sas27 and sas28 genes are involved in the biosynthesis of the cinnamoyl moiety in WS9326A.

Four thiol-oxidoreductases involved in the formation of disulphide bonds in the Streptomyces lividans TK21 secretory proteins.

BACKGROUND: Bacterial secretory proteins often require the formation of disulphide bonds outside the cell to acquire an active conformation. Thiol-disulphide oxidoreductases are enzymes that catalyse the formation of disulphide bonds. The bacterium Streptomyces lividans is a well-known host for the efficient secretion of overproduced homologous and heterologous secretory proteins of industrial application. Therefore, the correct conformation of these extracellular proteins is of great importance when engineering that overproduction. RESULTS: We have identified four acting thiol-disulphide oxidoreductases (TDORs) in S. lividans TK21, mutants in all TDOR candidates affect the secretion and activity of the Sec-dependent alpha-amylase, which contains several disulphide bonds, but the effect was more drastic in the case of the Sli-DsbA deficient strain. Thus, the four TDOR are required to obtain active alpha-amylase. Additionally, only mutations in Sli-DsbA and Sli-DsbB affect the secretion and activity of the Tat-dependent agarase, which does not form a disulphide bond, when it is overproduced. This suggests a possible role of the oxidised Sli-DsbA as a chaperone in the production of active agarase. CONCLUSIONS: Enzymes involved in the production of the extracellular mature active proteins are not fully characterised yet in Streptomyces lividans. Our results suggest that the role of thiol-disulphide oxidoreductases must be considered when engineering Streptomyces strains for the overproduction of homologous or heterologous secretory proteins of industrial application, irrespective of their secretion route, in order to obtain active, correctly folded proteins.

An Aromatic Dyad Motif in Dye Decolourising Peroxidases Has Implications for Free Radical Formation and Catalysis.

Dye decolouring peroxidases (DyPs) are the most recent class of heme peroxidase to be discovered. On reacting with H2 O2 , DyPs form a high-valent iron(IV)-oxo species and a porphyrin radical (Compound I) followed by stepwise oxidation of an organic substrate. In the absence of substrate, the ferryl species decays to form transient protein-bound radicals on redox active amino acids. Identification of radical sites in DyPs has implications for their oxidative mechanism with substrate. Using a DyP from Streptomyces lividans, referred to as DtpA, which displays low reactivity towards synthetic dyes, activation with H2 O2 was explored. A Compound I EPR spectrum was detected, which in the absence of substrate decays to a protein-bound radical EPR signal. Using a newly developed version of the Tyrosyl Radical Spectra Simulation Algorithm, the radical EPR signal was shown to arise from a pristine tyrosyl radical and not a mixed Trp/Tyr radical that has been widely reported in DyP members exhibiting high activity with synthetic dyes. The radical site was identified as Tyr374, with kinetic studies inferring that although Tyr374 is not on the electron-transfer pathway from the dye RB19, its replacement with a Phe does severely compromise activity with other organic substrates. These findings hint at the possibility that alternative electron-transfer pathways for substrate oxidation are operative within the DyP family. In this context, a role for a highly conserved aromatic dyad motif is discussed.

Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4.

Reinvestigation of the metabolite profile in a disruptant of the quinoprotein dehydrogenase (orf23) gene revealed that the Orf23 protein catalyzes dehydrogenation of the C23-C25 lactate moiety to pyruvate during lankacidin biosynthesis in Streptomyces rochei 7434AN4. The dehydrogenase activity was expressed and detected in a soluble fraction of the Streptomyces lividans recombinant harboring orf23. The Orf23 protein preferentially converts lankacidinol to lankacidin C in the presence of pyrroloquinoline quinone (PQQ). Other lankacidinol derivatives, lankacidinol A and iso-lankacidinol, were also converted to the corresponding C-24 keto compounds, lankacidin A (=sedecamycin) and iso-lankacidin C. Addition of various divalent metal cations, especially Ca2+, enhanced the dehydrogenase activity, whereas EDTA completely inhibited. These findings confirmed that the quinoprotein dehydrogenase Orf23 functions at the final oxidation step of lankacidin biosynthesis.

In Streptomyces lividans, acetyl-CoA synthetase activity is controlled by O-serine and Nɛ -lysine acetylation.

Protein acetylation is a rapid mechanism for control of protein function. Acetyl-CoA synthetase (AMP-forming, Acs) is the paradigm for the control of metabolic enzymes by lysine acetylation. In many bacteria, type I or II protein acetyltransferases acetylate Acs, however, in actinomycetes type III protein acetyltransferases control the activity of Acs. We measured changes in the activity of the Streptomyces lividans Acs (SlAcs) enzyme upon acetylation by PatB using in vitro and in vivo analyses. In addition to the acetylation of residue K610, residue S608 within the acetylation motif of SlAcs was also acetylated (PKTRSGK610 ). S608 acetylation rendered SlAcs inactive and non-acetylatable by PatB. It is unclear whether acetylation of S608 is enzymatic, but it was clear that this modification occurred in vivo in Streptomyces. In S. lividans, an NAD+ -dependent sirtuin deacetylase from Streptomyces, SrtA (a homologue of the human SIRT4 protein) was needed to maintain SlAcs function in vivo. We have characterized a sirtuin-dependent reversible lysine acetylation system in Streptomyces lividans that targets and controls the Acs enzyme of this bacterium. These studies raise questions about acetyltransferase specificity, and describe the first Acs enzyme in any organism whose activity is modulated by O-Ser and Nɛ -Lys acetylation.

Effects of Increased NADPH Concentration by Metabolic Engineering of the Pentose Phosphate Pathway on Antibiotic Production and Sporulation in Streptomyces lividans TK24.

Most of the biosynthetic pathways for secondary metabolites are influenced by carbon metabolism and supply of cytosolic NADPH. We engineered carbon distribution to the pentose phosphate pathway (PPP) and redesigned the host to produce high levels of NADPH and primary intermediates from the PPP. The main enzymes producing NADPH in the PPP, glucose 6-phosphate dehydrogenase (encoded by zwf1 and zwf2) and 6-phosphogluconate dehydrogenase (encoded by zwf3), were overexpressed with opc encoding a positive allosteric effector essential for Zwf activity in various combinations in Streptomyces lividans TK24. Most S. lividans transformants showed better cell growth and higher concentration of cytosolic NADPH than those of the control, and S. lividans TK24/pWHM3-Z23O2 containing zwf2+zwf3+opc2 showed the highest NADPH concentration but poor sporulation in R2YE medium. S. lividans TK24/pWHM3-Z23O2 in minimal medium showed the maximum growth (6.2 mg/ml) at day 4. Thereafter, a gradual decrease of biomass and a sharp increase of cytosolic NADPH and sedoheptulose 7-phosphate between days 2 and 4 and between days 1 and 3, respectively, were observed. Moreover, S. lividans TK24/pWHM3-Z23O2 produced 0.9 times less actinorhodin but 1.8 times more undecylprodigiosin than the control. These results suggested that the increased NADPH concentration and various intermediates from the PPP specifically triggered undecylprodigiosin biosynthesis that required many precursors and NADPH-dependent reduction reaction. This study is the first report on bespoke metabolic engineering of PPP routes especially suitable for producing secondary metabolites that need diverse primary precursors and NADPH, which is useful information for metabolic engineering in Streptomyces.

Kinetic characterisation of a dye decolourising peroxidase from Streptomyces lividans: new insight into the mechanism of anthraquinone dye decolourisation.

Dye decolourising peroxidases are the most recent family of haem peroxidases to be discovered. The oxidising potential of these enzymes is driven by the formation of ferryl intermediates that enables them to oxidise synthetic dye molecules that are widely used in the textile industry. We have investigated the catalytic cycle of a dye decolourising peroxidase (DtpA) from a biotechnologically important bacterium Streptomyces lividans. Using a combination of steady-state and stopped-flow kinetic investigations, we have determined the rate constants for all steps in the catalytic cycle with a range of substrate molecules. For most substrates, the value of kcat/Km measured by steady-state kinetics is equal to the slowest step in catalysis measured by stopped-flow spectroscopy, namely the decay of the ferryl FeIV[double bond, length as m-dash]O species (compound II) to form the ferric species. With the anthraquinone-based dye, reactive blue 19 (RB19) unusual steady-state kinetic behaviour is observed, which we propose through kinetic modelling of the catalytic cycle is due to a disproportionation mechanism of the dye. At low RB19 concentrations, the rate of disproportionation is slower than that of the rate determining step in DtpA, whereas at higher concentrations of RB19 the rate of disproportionation is faster. This mechanism obviates the need to postulate secondary sites for substrate binding on the enzyme which has been previously proposed for other dye decolourising haem peroxidases.

Active-site maturation and activity of the copper-radical oxidase GlxA are governed by a tryptophan residue.

GlxA from Streptomyces lividans is a mononuclear copper-radical oxidase and a member of the auxiliary activity family 5 (AA5). Its domain organisation and low sequence homology make it a distinct member of the AA5 family in which the fungal galactose 6-oxidase (Gox) is the best characterised. GlxA is a key cuproenzyme in the copper-dependent morphological development of S. lividans with a function that is linked to the processing of an extracytoplasmic glycan. The catalytic sites in GlxA and Gox contain two distinct one-electron acceptors comprising the copper ion and a 3'-(S-cysteinyl) tyrosine. The latter is formed post-translationally through a covalent bond between a cysteine and a copper-co-ordinating tyrosine ligand and houses a radical. In GlxA and Gox, a second co-ordination sphere tryptophan residue (Trp288 in GlxA) is present, but the orientation of the indole ring differs between the two enzymes, creating a marked difference in the π-π stacking interaction of the benzyl ring with the 3'-(S-cysteinyl) tyrosine. Differences in the spectroscopic and enzymatic activity have been reported between GlxA and Gox with the indole orientation suggested as a reason. Here, we report a series of in vivo and in vitro studies using the W288F and W288A variants of GlxA to assess the role of Trp288 on the morphology, maturation, spectroscopic and enzymatic properties. Our findings point towards a salient role for Trp288 in the kinetics of copper loading and maturation of GlxA, with its presence essential for stabilising the metalloradical site required for coupling catalytic activity and morphological development.

Another example of enzymatic promiscuity: the polyphosphate kinase of Streptomyces lividans is endowed with phospholipase D activity.

Polyphosphate kinases (PPK) from different bacteria, including that of Streptomyces lividans, were shown to contain the typical HKD motif present in phospholipase D (PLD) and showed structural similarities to the latter. This observation prompted us to investigate the PLD activity of PPK of S. lividans, in vitro. The ability of PPK to catalyze the hydrolysis of phosphatidylcholine (PC), the PLD substrate, was assessed by the quantification of [3H]phosphatidic acid (PA) released from [3H]PC-labeled ELT3 cell membranes. Basal cell membrane PLD activity as well as GTPγS-activated PLD activity was higher in the presence than in absence of PPK. After abolition of the basal PLD activity of the membranes by heat or tryptic treatment, the addition of PPK to cell membranes was still accompanied by an increased production of PA demonstrating that PPK also bears a PLD activity. PLD activity of PPK was also assessed by the production of choline from hydrolysis of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the presence of the Amplex Red reagent and compared to two commercial PLD enzymes. These data demonstrated that PPK is endowed with a weak but clearly detectable PLD activity. The question of the biological signification, if any, of this enzymatic promiscuity is discussed.

Improving the active expression of transglutaminase in Streptomyces lividans by promoter engineering and codon optimization.

BACKGROUND: Transglutaminases (TGase), which are synthesized as a zymogen (pro-TGase) in Streptomyces sp., are important enzymes in the food industry. Because this pro-peptide is essential for the correct folding of Streptomyces TGase, TGase is usually expressed in an inactive pro-TGase form, which is then converted to active TGase by the addition of activating proteases in vitro. In this study, Streptomyces hygroscopicus TGase was actively produced by Streptomyces lividans through promoter engineering and codon optimization. RESULTS: A gene fragment (tg1, 2.6 kb) that encoded the pro-TGase and its endogenous promoter region, signal peptide and terminator was amplified from S. hygroscopicus WSH03-13 and cloned into plasmid pIJ86, which resulted in pIJ86/tg1. After fermentation for 2 days, S. lividans TK24 that harbored pIJ86/tg1 produced 1.8 U/mL of TGase, and a clear TGase band (38 kDa) was detected in the culture supernatant. These results indicated that the pro-TGase was successfully expressed and correctly processed into active TGase in S. lividans TK24 by using the TGase promoter. Based on deletion analysis, the complete sequence of the TGase promoter is restricted to the region from -693 to -48. We also identified a negative element (-198 to -148) in the TGase promoter, and the deletion of this element increased the TGase production by 81.3 %, in contrast to the method by which S. lividans expresses pIJ86/tg1. Combining the deletion of the negative element of the promoter and optimization of the gene codons, the yield and productivity of TGase reached 5.73 U/mL and 0.14 U/mL/h in the recombinant S. lividans, respectively. CONCLUSIONS: We constructed an active TGase-producing strain that had a high yield and productivity, and the optimized TGase promoter could be a good candidate promoter for the expression of other proteins in Streptomyces.

Ligand Binding Enhances Millisecond Conformational Exchange in Xylanase B2 from Streptomyces lividans.

Xylanases catalyze the hydrolysis of xylan, an abundant carbon and energy source with important commercial ramifications. Despite tremendous efforts devoted to the catalytic improvement of xylanases, success remains limited because of our relatively poor understanding of their molecular properties. Previous reports suggested the potential role of atomic-scale residue dynamics in modulating the catalytic activity of GH11 xylanases; however, dynamics in these studies was probed on time scales orders of magnitude faster than the catalytic time frame. Here, we used nuclear magnetic resonance titration and relaxation dispersion experiments ((15)N-CPMG) in combination with X-ray crystallography and computational simulations to probe conformational motions occurring on the catalytically relevant millisecond time frame in xylanase B2 (XlnB2) and its catalytically impaired mutant E87A from Streptomyces lividans 66. Our results show distinct dynamical properties for the apo and ligand-bound states of the enzymes. The apo form of XlnB2 experiences conformational exchange for residues in the fingers and palm regions of the catalytic cleft, while the catalytically impaired E87A variant displays millisecond dynamics only in the fingers, demonstrating the long-range effect of the mutation on flexibility. Ligand binding induces enhanced conformational exchange of residues interacting with the ligand in the fingers and thumb loop regions, emphasizing the potential role of residue motions in the fingers and thumb loop regions for recognition, positioning, processivity, and/or stabilization of ligands in XlnB2. To the best of our knowledge, this work represents the first experimental characterization of millisecond dynamics in a GH11 xylanase family member. These results offer new insights into the potential role of conformational exchange in GH11 enzymes, providing essential dynamic information to help improve protein engineering and design applications.