Characterization of the cystargolide biosynthetic gene cluster and functional analysis of the methyltransferase CysG.

Cystargolides are natural products originally isolated from Kitasatospora cystarginea NRRL B16505 as inhibitors of the proteasome. They are composed of a dipeptide backbone linked to a ß-lactone warhead. Recently, we identified the cystargolide biosynthetic gene cluster, but systematic genetic analyses had not been carried out because of the lack of a heterologous expression system. Here, we report the discovery of a homologous cystargolide biosynthetic pathway in Streptomyces durhamensis NRRL-B3309 by genome mining. The gene cluster was cloned via transformation-associated recombination and heterologously expressed in Streptomyces coelicolor M512. We demonstrate that it contains all genes necessary for the production of cystargolide A and B. Single gene deletion experiments reveal that only five of the eight genes from the initially proposed gene cluster are essential for cystargolide synthesis. Additional insights into the cystargolide pathway could be obtained from in vitro assays with CysG and chemical complementation of the respective gene knockout. This could be further supported by the in vitro investigation of the CysG homolog BelI from the belactosin biosynthetic gene cluster. Thereby, we confirm that CysG and BelI catalyze a cryptic SAM-dependent transfer of a methyl group that is critical for the construction of the cystargolide and belactosin ß-lactone warheads.

Four amino acids define the CO2 binding pocket of enoyl-CoA carboxylases/reductases.

Carboxylases are biocatalysts that capture and convert carbon dioxide (CO2) under mild conditions and atmospheric concentrations at a scale of more than 400 Gt annually. However, how these enzymes bind and control the gaseous CO2 molecule during catalysis is only poorly understood. One of the most efficient classes of carboxylating enzymes are enoyl-CoA carboxylases/reductases (Ecrs), which outcompete the plant enzyme RuBisCO in catalytic efficiency and fidelity by more than an order of magnitude. Here we investigated the interactions of CO2 within the active site of Ecr from Kitasatospora setae Combining experimental biochemistry, protein crystallography, and advanced computer simulations we show that 4 amino acids, N81, F170, E171, and H365, are required to create a highly efficient CO2-fixing enzyme. Together, these 4 residues anchor and position the CO2 molecule for the attack by a reactive enolate created during the catalytic cycle. Notably, a highly ordered water molecule plays an important role in an active site that is otherwise carefully shielded from water, which is detrimental to CO2 fixation. Altogether, our study reveals unprecedented molecular details of selective CO2 binding and C-C-bond formation during the catalytic cycle of nature's most efficient CO2-fixing enzyme. This knowledge provides the basis for the future development of catalytic frameworks for the capture and conversion of CO2 in biology and chemistry.

Analysis of oscillatory rocking curve by dynamical diffraction in protein crystals.

High-quality protein crystals meant for structural analysis by X-ray diffraction have been grown by various methods. The observation of dynamical diffraction in protein crystals is an interesting topic because dynamical diffraction generally occurs in perfect crystals such as Si crystals. However, to our knowledge, there is no report yet on protein crystals showing clear dynamical diffraction. We wonder whether the perfection of protein crystals might still be low compared with that of high-quality Si crystals. Here, we present observations of the oscillatory profile of rocking curves for protein crystals such as glucose isomerase crystals. The oscillatory profiles are in good agreement with those predicted by the dynamical theory of diffraction. We demonstrate that dynamical diffraction occurs even in protein crystals. This suggests the possibility of the use of dynamical diffraction for the determination of the structure and charge density of proteins.

Versatile Oxidase and Dehydrogenase Activities of Bacterial Pyranose 2-Oxidase Facilitate Redox Cycling with Manganese Peroxidase In Vitro.

Pyranose 2-oxidase (POx) has long been accredited a physiological role in lignin degradation, but evidence to provide insights into the biochemical mechanisms and interactions is insufficient. There are ample data in the literature on the oxidase and dehydrogenase activities of POx, yet the biological relevance of this duality could not be established conclusively. Here we present a comprehensive biochemical and phylogenetic characterization of a novel pyranose 2-oxidase from the actinomycetous bacterium Kitasatospora aureofaciens (KaPOx) as well as a possible biomolecular synergism of this enzyme with peroxidases using phenolic model substrates in vitro A phylogenetic analysis of both fungal and bacterial putative POx-encoding sequences revealed their close evolutionary relationship and supports a late horizontal gene transfer of ancestral POx sequences. We successfully expressed and characterized a novel bacterial POx gene from K. aureofaciens, one of the putative POx genes closely related to well-known fungal POx genes. Its biochemical characteristics comply with most of the classical hallmarks of known fungal pyranose 2-oxidases, i.e., reactivity with a range of different monosaccharides as electron donors as well as activity with oxygen, various quinones, and complexed metal ions as electron acceptors. Thus, KaPOx shows the pronounced duality of oxidase and dehydrogenase similar to that of fungal POx. We further performed efficient redox cycling of aromatic lignin model compounds between KaPOx and manganese peroxidase (MnP). In addition, we found a Mn(III) reduction activity in KaPOx, which, in combination with its ability to provide H2O2, implies this and potentially other POx as complementary enzymatic tools for oxidative lignin degradation by specialized peroxidases.IMPORTANCE Establishment of a mechanistic synergism between pyranose oxidase and (manganese) peroxidases represents a vital step in the course of elucidating microbial lignin degradation. Here, the comprehensive characterization of a bacterial pyranose 2-oxidase from Kitasatospora aureofaciens is of particular interest for several reasons. First, the phylogenetic analysis of putative pyranose oxidase genes reveals a widespread occurrence of highly similar enzymes in bacteria. Still, there is only a single report on a bacterial pyranose oxidase, stressing the need of closing this gap in the scientific literature. In addition, the relatively small K. aureofaciens proteome supposedly supplies a limited set of enzymatic functions to realize lignocellulosic biomass degradation. Both enzyme and organism therefore present a viable model to study the mechanisms of bacterial lignin decomposition, elucidate physiologically relevant interactions with specialized peroxidases, and potentially realize biotechnological applications.

Diversity of family GH46 chitosanases in Kitasatospora setae KM-6054.

The genome of Kitasatospora setae KM-6054, a soil actinomycete, has three genes encoding chitosanases belonging to GH46 family. The genes (csn1-3) were cloned in Streptomyces lividans and the corresponding enzymes were purified from the recombinant cultures. The csn2 clone yielded two proteins (Csn2BH and Csn2H) differing by the presence of a carbohydrate-binding domain. Sequence analysis showed that Csn1 and Csn2H were canonical GH46 chitosanases, while Csn3 resembled chitosanases from bacilli. The activity of the four chitosanases was tested in a variety of conditions and on diverse chitosan forms, including highly N-deacetylated chitosan or chitosan complexed with humic or polyphosphoric acid. Kinetic parameters were also determined. These tests unveiled the biochemical diversity among these chitosanases and the peculiarity of Csn3 compared with the other three enzymes. The observed biochemical diversity is discussed based on structural 3D models and sequence alignment. This is a first study of all the GH46 chitosanases produced by a single microbial strain.

A rifamycin inactivating phosphotransferase family shared by environmental and pathogenic bacteria.

Many environmental bacteria are multidrug-resistant and represent a reservoir of ancient antibiotic resistance determinants, which have been linked to genes found in pathogens. Exploring the environmental antibiotic resistome, therefore, reveals the diversity and evolution of antibiotic resistance and also provides insight into the vulnerability of clinically used antibiotics. In this study, we describe the identification of a highly conserved regulatory motif, the rifampin (RIF) -associated element (RAE), which is found upstream of genes encoding RIF-inactivating enzymes from a diverse collection of actinomycetes. Using gene expression assays, we confirmed that the RAE is involved in RIF-responsive regulation. By using the RAE as a probe for new RIF-associated genes in several actinomycete genomes, we identified a heretofore unknown RIF resistance gene, RIF phosphotransferase (rph). The RPH enzyme is a RIF-inactivating phosphotransferase and represents a new protein family in antibiotic resistance. RPH orthologs are widespread and found in RIF-sensitive bacteria, including Bacillus cereus and the pathogen Listeria monocytogenes. Heterologous expression and in vitro enzyme assays with purified RPHs from diverse bacterial genera show that these enzymes are capable of conferring high-level resistance to a variety of clinically used rifamycin antibiotics. This work identifies a new antibiotic resistance protein family and reinforces the fact that the study of resistance in environmental organisms can serve to identify resistance elements with relevance to pathogens.

Mechanistic Investigations of Two Bacterial Diterpene Cyclases: Spiroviolene Synthase and Tsukubadiene Synthase.

The mechanisms of two diterpene cyclases from streptomycetes-one with an unknown product that was identified as the spirocyclic hydrocarbon spiroviolene and one with the known product tsukubadiene-were investigated in detail by isotope labeling experiments. Although the structures of the products were very different, the cyclization mechanisms of both enzymes proceed through the same initial cyclization reactions, before they diverge towards the individual products, which is reflected in the close phylogenetic relationship of the enzymes.

Characterization and gene cloning of a maltotriose-forming exo-amylase from Kitasatospora sp. MK-1785.

A maltotriose-forming amylase (G3Amy) from Kitasatospora sp. MK-1785 was successfully isolated from a soil sample by inhibiting typical extracellular α-amylases using a proteinaceous α-amylase inhibitor. G3Amy was purified from the MK-1785 culture supernatant and characterized. G3Amy produced maltotriose as the principal product from starch and was categorized as an exo-α-amylase. G3Amy could also transfer maltotriose to phenolic and alcoholic compounds. Therefore, G3Amy can be useful for not only maltotriose manufacture but also maltooligosaccharide-glycoside synthesis. Further, the G3Amy gene was cloned and expressed in Escherichia coli cells. Analysis of its deduced amino acid sequence revealed that G3Amy consisted of an N-terminal GH13 catalytic domain and two C-terminal repeat starch-binding domains belonging to CBM20. It is suggested that natural G3Amy was subjected to proteolysis at N-terminal region of the anterior CBM20 in the C-terminal region. As with natural G3Amy, recombinant G3Amy could produce and transfer maltotriose from starch.

Structures and Biosynthesis of Corvol Ethers--Sesquiterpenes from the Actinomycete Kitasatospora setae.

Here we present the functional characterization of a sesquiterpene cyclase from Kitasatospora setae. The enzyme converts the sesquiterpene precursor farnesyl diphosphate (FPP) into two previously unknown and unstable sesquiterpene ethers for which we propose the trivial names corvol ethers A and B. Both compounds were purified and their structures were determined by one- and two-dimensional NMR spectroscopy. A biosynthetic mechanism for the FPP cyclization by the corvol ether synthase was proposed. The results from the incubation experiments of the corvol ether synthase with isotopically labeled precursors were in line with this mechanism, while alternative mechanisms could clearly be ruled out.

Hedycaryol synthase in complex with nerolidol reveals terpene cyclase mechanism.

The biosynthesis of terpenes is catalysed by class I and II terpene cyclases. Here we present structural data from a class I hedycaryol synthase in complex with nerolidol, serving as a surrogate for the reaction intermediate nerolidyl diphosphate. This prefolded ligand allows mapping of the active site and hence the identification of a key carbonyl oxygen of Val179, a highly conserved helix break (G1/2) and its corresponding helix dipole. Stabilising the carbocation at the substrate's C1 position, these elements act in concert to catalyse the 1,10 ring closure, thereby exclusively generating the anti-Markovnikov product. The delineation of a general mechanistic scaffold was confirmed by site-specific mutations. This work serves as a basis for understanding carbocation chemistry in enzymatic reactions and should contribute to future application of these enzymes in organic synthesis.

In vitro phosphinate methylation by PhpK from Kitasatospora phosalacinea.

Radical S-adenosyl-L-methionine, cobalamin-dependent methyltransferases have been proposed to catalyze the methylations of unreactive carbon or phosphorus atoms in antibiotic biosynthetic pathways. To date, none of these enzymes has been purified or shown to be active in vitro. Here we demonstrate the activity of the P-methyltransferase enzyme, PhpK, from the phosalacine producer Kitasatospora phosalacinea. PhpK catalyzes the transfer of a methyl group from methylcobalamin to 2-acetylamino-4-hydroxyphosphinylbutanoate (N-acetyldemethylphosphinothricin) to form 2-acetylamino-4-hydroxymethylphosphinylbutanoate (N-acetylphosphinothricin). This transformation gives rise to the only carbon-phosphorus-carbon linkage known to occur in nature.

Swelling, mechanical, and barrier properties of albedo-based films prepared in the presence of phaseolin cross-linked or not by transglutaminase.

Edible films were obtained from Citrus paradisi grapefruit albedo homogenates and bean protein phaseolin modified or not by the enzyme transglutaminase. Swelling capability, barrier performance to water vapor, oxygen and carbon dioxide, and mechanical properties of such films were investigated. The addition of the protein, mostly in the presence of transglutaminase, provide films less swellable at pH values above 5 compared to films made by albedo homogenates only, whereas the action of the enzyme clearly improves mechanical properties producing more stretchable and elastic films. Moreover, transglutaminase-mediated cross-linking of phaseolin gives rise to films less permeable to carbon dioxide and able to offer a high barrier to water vapor. These findings suggest that albedo-phaseolin film prepared in the presence of transglutaminase can be a promising candidate to be used as food edible wrap.

GH-10 and GH-11 Endo-1,4-ß-xylanase enzymes from Kitasatospora sp. produce xylose and xylooligosaccharides from sugarcane bagasse with no xylose inhibition.

A novel strategy for the low-cost, high-yield co-production of xylose and xylooligosaccharides together with no xylose inhibition was developed using a novel heterologous expression of XYN10Ks_480 endo-1,4-ß-xylanase with a ricin-type ß-trefoil type of domain and XYN11Ks_480 endo-1,4-ß-xylanase with a CBM 2 superfamily from the Kitasatospora sp in an actinomycetes expression system. Xylose is the main building block for hemicellulose xylan. Our findings demonstrated high levels of expression and catalytic activity for XYN10Ks_480 during hydrolysis of the extracted xylan of bagasse, and three types of xylan-based substrates were used to produce xylose and xylooligosaccharides. However, hydrolysis by XYN11Ks_480 produced xylooligosaccharides without xylose formation. This study demonstrated how integrating sodium hypochlorite-extracted xylan and enzymatic hydrolysis could provide an alternative strategy for the generation of XOS from lignocellulosic material.

Discovery of a Short-Chain ε-Poly-l-lysine and Its Highly Efficient Production via Synthetase Swap Strategy.

ε-Poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide, which is generally recognized as safe for use as a food preservative. To date, the production capacity of strains that produce low-molecular weight ε-PL remains very low and thus unsuitable for industrial production. Here, we report a new low-molecular weight ε-PL-producing Kitasatospora aureofaciens strain. The ε-PL synthase gene of this strain was cloned into a high ε-PL-producing Streptomyces albulus strain. The resulting recombinant strain efficiently produced ε-PL with a molecular weight of 1.3-2.3 kDa and yielded of 23.6 g/L following fed-batch fermentation in a 5 L bioreactor. In addition, circular dichroism spectra showed that this ε-PL takes on a conformation similar to an antiparallel pleated-sheet. Moreover, it demonstrated better antimicrobial activity against yeast compared to the 3.2-4.5 kDa ε-PL. This study provides a highly efficient strategy for production of the low-molecular weight ε-PL, which helps to expand its potential applications.

Remarkable enhancement in PLD activity from Streptoverticillium cinnamoneum by substituting serine residue into the GG/GS motif.

The gene that encodes phospholipase D (PLD) from Streptoverticillium cinnamoneum contains three consensus regions (Region I, II and IV as shown in Fig. 1A) that are conserved among the PLD superfamily. The glycine-glycine (GG) motif in Region I and the glycine-serine (GS) motif in Region IV are also conserved in the PLD superfamily. These (GG and GS) motifs are located 7 residues downstream from each HKD motif. In an investigation of fifteen GG/GS motif mutants, generated as fusion proteins with maltose-binding protein (MBP-PLDs), three highly active mutants were identified. Three of the mutants (G215S, G216S, and G216S-S489G) contained a serine residue in the GG motif, and exhibited approximately a 9-27-fold increased transphosphatidylation activity to DPPC compared with recombinant wild type MBP-PLD. When heat stability was compared between three mutants and the recombinant wild type, only G216S-S489G showed heat labile properties. It appears that the 489th serine residue in the GS motif also contributes to the thermal stability of the enzyme. In addition, the GG/GS motif was very close to the active center residue, including two HKD motifs, as shown by computer modeling. The findings suggest that the GG/GS motif of PLD is a key motif that affects catalytic function and enzymatic stability.

Biosynthesis of 2-amino-3-hydroxycyclopent-2-enone moiety of bafilomycin in Kitasatospora cheerisanensis KCTC2395.

Bafilomycins produced by Kitasatospora cheerisanensis KCTC- 2395 belong to the 16-membered macrolactone family plecomacrolide antibiotics. Bafilomycin B1 contains 2-amino- 3-hydroxycyclopent-2-enone (C5N), a five membered ring, which gets condensed via an amide linkage to bafilomycin polyketide. To study the biosynthetic pathway of C5N during bafilomycin biosynthesis in K. cheerisanensis KCTC2395, we attempted the functional analysis of two putative genes, encoding 5-aminolevulinic acid synthase (ALAS) and acyl- CoA ligase (ACL). The amplified putative genes for ALAS and ACL were cloned into the E. coli expression vector pET- 32a(+) plasmid, following which the soluble recombinant ALAS and ACL proteins were purified through nickel-affinity column chromatography. Through HPLC analysis of the enzyme reaction mixture, we confirmed the products of putative ALAS and ACL reaction as 5-aminolevulinic acid (5-ALA) and 5-ALA-CoA, respectively. The optimal pH for the putative ALAS reaction was 7.5, and for putative ACL reaction was 7.0, as confirmed by the colorimetric assay. Furthermore, pyridoxal 5'-phosphate (PLP) was found to be an essential cofactor in the putative ALAS reaction, and ATP was a cofactor for the putative ACL catalysis. Finally, we also confirmed that the simultaneous treatment of putative ACL and putative ALAS enzymes resulted in the production of C5N compound from 5-ALA.

Biochemical characterisation of recombinant Streptomyces pristinaespiralis L-lysine cyclodeaminase.

L-Lysine cyclodeaminase from Streptomyces pristinaespiralis was heterologously expressed in Escherichia coli, isolated to 90% purity after two purification steps and characterised. The size of the isolated recombinant enzyme was in agreement with the theoretical size calculated from the corresponding gene. We demonstrated that our preparation converts L-lysine to L-pipecolic acid (enantiomeric excess >95%) after isolating and identifying the conversion product by LC/MS, NMR and IR. This conversion followed Michaelis-Menten kinetics with a K(m) of 1.39+/-0.32 mM. The enzyme activity was maximal at pH 6.7. Reducing conditions, the presence of glycerol and in particular the presence of iron(II) significantly enhanced the L-lysine cyclodeaminase activity. Although the heat stability of the enzyme diminished significantly after 37 degrees C, the initial rate of reaction was maximal at 61 degrees C. We found no requirement for an external cofactor for full activity, although sequence data indicate NAD+ as cofactor. Upon enzyme denaturation, NAD+ release was observed, which indicates very tight binding of NAD+ to the enzyme. In parallel we developed selection and screening assays for lysine cyclodeaminase, which we adapted to microtitre plate format and validated. Among twenty-eight lysine analogues screened for turnover/binding to the enzyme, three were identified as substrates (L-ornithine, 5-hydroxylysine and L-4-thialysine), while another six (4-azalysine, L-2,4-diaminobutyric acid, 1,5-diaminopentane, N-epsilon-trifluoroacetyl-L-lysine, N-epsilon-Boc-L-lysine and N-epsilon-methyl-L-lysine) were shown to compete against L-lysine turnover without being converted by the enzyme. All substrates displayed Michaelis-Menten kinetics upon turnover by lysine cyclodeaminase. Our results indicate that the lysine cyclodeaminase from Streptomyces pristinaespiralis is a highly enantioselective enzyme at the substrate recognition and conversion levels, in both cases in favour of the l-isomer.

Substrate specificity of streptomyces transglutaminases.

Transglutaminase (TGase) is a multifunctional enzyme vital for many physiologic processes, such as cell differentiation, tissue regeneration, and plant pathogenicity. The acyl transfer function of the enzyme can activate primary amines and, consequently, attach them onto a peptidyl glutamine, a reaction important for various in vivo and in vitro protein crosslinking and modification processes. To understand better the structure-function relationship of the enzyme and to develop it further as an industrial biocatalyst, we studied TGase secreted by several Streptomyces species and Phytophthora cactorum. We purified the enzyme from S. lydicus, S. platensis, S. nigrescens, S. cinnamoneus, and S. hachijoensis. The pH and temperature profiles of S. lydicus, S. platensis, and S. nigrescens TGases were determined. The specificity of S. lydicus TGase toward its acyl-accepting amine substrates was characterized. Correlation of the electronic and steric features of the substrates with their reactivity supported the mechanism previously proposed for Streptomyces mobaraensis TGase.

Novel hemostatic agents based on gelatin-microbial transglutaminase mix.

Hemostasis is a major challenge in surgical procedures and traumas. Conventional hemostatic methods have limited efficacy and may cause additional tissue damage. In this study, we designed a novel hemostatic agent based on the in situ gel formation of gelatin cross-linked by a novel microbial transglutaminase (mTGase), in which the amino acid sequences differed from commercial mTGases. The new hemostatic agent showed the same biochemical crosslinking chemistry as the final stages of the blood coagulation cascade while using gelatin as a "structural" protein (rather than fibrin) and a calcium-independent mTGase as the crosslinking catalyst (rather than factor XIIIa). In rat liver hemostasis models, the hemostatic agent not only showed a similar hemostatic effect as that of SURGIFLO® (positive control), but also stronger adhesion strength and elasticity than SURGIFLO®. Therefore, this biomimetic gelatin-mTGase mix hemostatic is a novel and effective surgical sealant.

Streptomyces virginiae PPDC Is a New Type of Phenylpyruvate Decarboxylase Composed of Two Subunits.

Streptomyces virginiae phenylpyruvate decarboxylase (PPDC) has not been identified before. Two putative branched-chain α-keto acid dehydrogenase subunit genes bkdC and bkdD from S. virginiae are similar to halves of other PPDC coding sequences. We cloned and characterized them biochemically in this work. The two proteins formed a stable complex attested by pull-down assay, consistent with the finding that their soluble expression was obtained only when they were coexpressed in Escherichia coli. The subunits were redesignated as SvPPDCα and SvPPDCß, because the SvPPDCα/ß complex catalyzed the conversion of phenylpyruvate to phenylacetaldehyde, reflecting the nature of the enzyme. Moreover, mutations of conserved residues in either of the two subunits led to inactivation or decreased specific activity of the enzymatic reaction. All previously identified PPDCs are encoded by a single gene. Here, we identified a new type of PPDC that contains two subunits, which gives new insights into the PPDC family.