substrate specificities
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2021 ◽  
Author(s):  
Marina Borisova ◽  
Katja Balbuchta ◽  
Andrew Lovering ◽  
Alexander Titz ◽  
Christoph Mayer

ABSTRACTThe Gram-negative periodontal pathogen Tannerella forsythia is inherently auxotrophic for N-acetylmuramic acid (MurNAc), which is an essential carbohydrate constituent of the peptidoglycan (PGN) of the bacterial cell wall. Thus, to build up its cell wall, T. forsythia strictly depends on the salvage of exogenous MurNAc or sources of MurNAc, such as polymeric or fragmentary PGN, derived from cohabiting bacteria within the oral microbiome. In our effort to elucidate how T. forsythia satisfies its demand for MurNAc, we recognized that the organism possesses three putative orthologs of the exo-β-N-acetylmuramidase BsNamZ from Bacillus subtilis, which cleaves non-reducing end, terminal MurNAc entities from the artificial substrate pNP-MurNAc and the naturally-occurring disaccharide substrate MurNAc-β-1,4-N-acetylglucosamine (GlcNAc). TfNamZ1 and TfNamZ2 were successfully purified as soluble, pure recombinant His6-fusions and characterized as exo-lytic β-N-acetylmuramidases with distinct substrate specificities. The activity of TfNamZ1 was considerably lower compared to TfNamZ2 and BsNamZ, in the cleavage of pNP-MurNAc and MurNAc-GlcNAc. When peptide-free PGN glycans were used as substrates, we revealed striking differences in the specificity and mode of action of these enzymes, as analyzed by mass spectrometry. TfNamZ1, but not TfNamZ2 or BsNamZ, released GlcNAc-MurNAc disaccharides from these glycans. In addition, glucosamine (GlcN)-MurNAc disaccharides were generated when partially N-deacetylated PGN glycans from B. subtilis 168 were applied. This characterizes TfNamZ1 as a unique disaccharide-forming exo-lytic β-N-acetylmuramidase (exo-disaccharidase), and, TfNamZ2 and BsNamZ as sole MurNAc monosaccharide-lytic exo-β-N-acetylmuramidases.IMPORTANCETwo exo-β-N-acetylmuramidases from T. forsythia belonging to glycosidase family GH171 (www.cazy.org) were shown to differ in their activities, thus revealing a functional diversity within this family: NamZ1 releases disaccharides (GlcNAc-MurNAc/GlcN-MurNAc) from the non-reducing ends of PGN glycans, whereas NamZ2 releases terminal MurNAc monosaccharides. This work provides a better understanding of how T. forsythia may acquire the essential growth factor MurNAc by the salvage of PGN from cohabiting bacteria in the oral microbiome, which may pave avenues for the development of anti-periodontal drugs. On a broad scale, our study indicates that the utilization of PGN as a nutrient source, involving exo-lytic N-acetylmuramidases with different modes of action, appears to be a general feature of bacteria, particularly among the phylum Bacteroidetes.


2021 ◽  
Author(s):  
◽  
Katherine Robins

<p>Non-ribosomal peptide synthetases (NRPSs) are multi-modular biosynthetic enzymes that are responsible for the production of many bioactive secondary metabolites produced by microorganisms. They are activated by phosphopantetheinyl transferase (PPTase) enzymes, which attach an essential prosthetic group to a specific site within a “carrier protein” (CP) domain that is an integral part of each NRPS module. Of particular importance in this work is the NRPS BpsA, which produces a blue pigment called indigoidine; but only when BpsA has first been activated by a PPTase. BpsA can be used as a reporter for PPTase activity, to identify PPTases and/or measure their activity. Several CP-substituted BpsA variants were used, in order to study and identify PPTases which may recognise different CP domains. The first part of the research described in this thesis examined the features of foreign CP interactions within BpsA that made these functional substitutions possible. Two key residues, the +4 and +24 positions relative to an invariant serine, were found to be highly important; with appropriate substitutions at these positions yielding active CP-substituted variants.  Wild type BpsA and the CP-substituted variants were then used as the basis of a screen to discover new PPTase genes, and associated natural product biosynthetic genes, from metagenomic libraries. The vast majority of bacteria that produce bioactive secondary metabolites are unable to be cultured under laboratory conditions; screening metagenomic libraries is a way to access this untapped biodiversity in order to discover new natural products. Two environmental DNA libraries were screened, and PPTase genes were identified via their ability to activate BpsA, giving rise to blue colonies in high throughput agar plate screens. This screen proved to be a powerful enrichment strategy with almost half of the novel 21 PPTase genes recovered also linked to biosynthetic gene clusters. Using the evolved CP-substituted BpsA variants (and thereby altering the PPTase recognition site) enabled a wider variety of hits to be found. This led to the hypothesis that some of the PPTases discovered via this screening method would have non-overlapping substrate specificities, a beneficial property for certain PPTase applications.  The 21 PPTase genes discovered via metagenomic screening were characterised further, using a series of assays involving BpsA to measure their activity. As is common for PPTase enzymes, there were difficulties in obtaining enough soluble protein via purification to perform a detailed analysis of each. Those that were able to be purified had much lower activity than other previously characterised PPTases, and were also not as specific for their CP substrates as they had first appeared to be. Due to these low activity levels, several other previously characterised PPTases were also studied further using the BpsA methods. All PPTases showed a relatively broad activity across a range of CP substrates.  The desire to obtain PPTases with more specific substrate specificities led to the development of a directed evolution screen to alter PPTase CP specificity. In a proof-of-principle study the E. coli PPTase EntD was evolved to lose activity with the BpsA CP while retaining activity with its native CP. This screen, the first of its kind to evolve PPTases for greater CP substrate specificity, was successful in recovering several improved variants. These variants had either completely abolished or vastly decreased activity for the WT BpsA CP while retaining the ability to activate the native (EntF) CP domain. The general strategy developed here can be applied to the evolution of other PPTases and CP substrates.</p>


2021 ◽  
Author(s):  
◽  
Katherine Robins

<p>Non-ribosomal peptide synthetases (NRPSs) are multi-modular biosynthetic enzymes that are responsible for the production of many bioactive secondary metabolites produced by microorganisms. They are activated by phosphopantetheinyl transferase (PPTase) enzymes, which attach an essential prosthetic group to a specific site within a “carrier protein” (CP) domain that is an integral part of each NRPS module. Of particular importance in this work is the NRPS BpsA, which produces a blue pigment called indigoidine; but only when BpsA has first been activated by a PPTase. BpsA can be used as a reporter for PPTase activity, to identify PPTases and/or measure their activity. Several CP-substituted BpsA variants were used, in order to study and identify PPTases which may recognise different CP domains. The first part of the research described in this thesis examined the features of foreign CP interactions within BpsA that made these functional substitutions possible. Two key residues, the +4 and +24 positions relative to an invariant serine, were found to be highly important; with appropriate substitutions at these positions yielding active CP-substituted variants.  Wild type BpsA and the CP-substituted variants were then used as the basis of a screen to discover new PPTase genes, and associated natural product biosynthetic genes, from metagenomic libraries. The vast majority of bacteria that produce bioactive secondary metabolites are unable to be cultured under laboratory conditions; screening metagenomic libraries is a way to access this untapped biodiversity in order to discover new natural products. Two environmental DNA libraries were screened, and PPTase genes were identified via their ability to activate BpsA, giving rise to blue colonies in high throughput agar plate screens. This screen proved to be a powerful enrichment strategy with almost half of the novel 21 PPTase genes recovered also linked to biosynthetic gene clusters. Using the evolved CP-substituted BpsA variants (and thereby altering the PPTase recognition site) enabled a wider variety of hits to be found. This led to the hypothesis that some of the PPTases discovered via this screening method would have non-overlapping substrate specificities, a beneficial property for certain PPTase applications.  The 21 PPTase genes discovered via metagenomic screening were characterised further, using a series of assays involving BpsA to measure their activity. As is common for PPTase enzymes, there were difficulties in obtaining enough soluble protein via purification to perform a detailed analysis of each. Those that were able to be purified had much lower activity than other previously characterised PPTases, and were also not as specific for their CP substrates as they had first appeared to be. Due to these low activity levels, several other previously characterised PPTases were also studied further using the BpsA methods. All PPTases showed a relatively broad activity across a range of CP substrates.  The desire to obtain PPTases with more specific substrate specificities led to the development of a directed evolution screen to alter PPTase CP specificity. In a proof-of-principle study the E. coli PPTase EntD was evolved to lose activity with the BpsA CP while retaining activity with its native CP. This screen, the first of its kind to evolve PPTases for greater CP substrate specificity, was successful in recovering several improved variants. These variants had either completely abolished or vastly decreased activity for the WT BpsA CP while retaining the ability to activate the native (EntF) CP domain. The general strategy developed here can be applied to the evolution of other PPTases and CP substrates.</p>


2021 ◽  
Author(s):  
Souradeep Basu ◽  
Paul Nurse ◽  
Andrew Jones

Abstract Cyclin dependent kinases (CDKs) lie at the heart of eukaryotic cell cycle control, with different Cyclin-CDK complexes initiating DNA replication (S-CDKs) and mitosis (M-CDKs). However, the principles on which Cyclin-CDKs organise the temporal order of cell cycle events are contentious. The currently most widely accepted model, is that the S-CDKs and M-CDKs are functionally specialised, with significant different substrate specificities to execute different cell cycle events. A second model is that S-CDKs and M-CDKs are redundant with each other, with both acting as sources of overall cellular CDK activity. Here we reconcile these two views of core cell cycle control. Using a multiplexed phosphoproteomics assay of in vivo S-CDK and M-CDK activities in fission yeast, we show that S-CDK and M-CDK substrate specificities are very similar, showing that S-CDKs are not completely specialised for S-phase alone. Normally S-CDK cannot undergo mitosis, but is able to do so when Protein Phosphatase 1 (PP1) is removed from the centrosome, allowing several mitotic substrates to be better phosphorylated by S-CDK in vivo. Thus, an increase in S-CDK activity in vivo is sufficient to allow S-CDK to carry out M-CDK function. Therefore, we unite the two opposing views of cell cycle control, showing that the core cell cycle engine which temporally orders cell cycle progression is largely based upon a quantitative increase of CDK activity through the cell cycle, combined with minor qualitative differences in catalytic specialisation of S-CDKs and M-CDKs.


2021 ◽  
Vol 7 (10) ◽  
pp. 873
Author(s):  
Sudarma Dita Wijayanti ◽  
Leander Sützl ◽  
Adèle Duval ◽  
Dietmar Haltrich

The CAZy auxiliary activity family 3 (AA3) comprises FAD-dependent enzymes belonging to the superfamily of glucose-methanol-choline (GMC) oxidoreductases. Glucose oxidase (GOx; EC 1.1.3.4) and glucose dehydrogenase (GDH; EC 1.1.5.9) are part of subfamily AA3_2 and catalyze the oxidation of β-D-glucose at its anomeric carbon to D-glucono-1,5-lactone. Recent phylogenetic analysis showed that AA3_2 glucose oxidoreductases can be grouped into four major clades, GOx I and GDH I–III, and in minor clades such as GOx II or distinct subclades. This wide sequence space of AA3_2 glucose oxidoreductases has, however, not been studied in detail, with mainly members of GOx I and GDH I studied biochemically or structurally. Here, we report the biochemical characterization of four fungal glucose oxidoreductases from distinct, hitherto unexplored clades or subclades. The enzyme from Aureobasidium subglaciale, belonging to the minor GOx II clade, showed a typical preference for oxygen and glucose, confirming the correct annotation of this clade. The other three enzymes exhibited strict dehydrogenase activity with different substrate specificities. GDH II from Trichoderma virens showed an almost six-fold higher catalytic efficiency for maltose compared to glucose. The preferred substrate for the two GDH III enzymes from Rhizoctonia solani and Ustilago maydis was gentiobiose, a β(1→6) disaccharide, as judged from the catalytic efficiency. Overall, the newly studied AA3_2 glucose oxidoreductases showed a much broader substrate spectrum than the archetypal GOx from Aspergillus niger, which belongs to clade GOx I.


Molecules ◽  
2021 ◽  
Vol 26 (18) ◽  
pp. 5693
Author(s):  
Juan Huang ◽  
Qin Xu ◽  
Zhuo Liu ◽  
Nitin Jain ◽  
Madhusudan Tyagi ◽  
...  

Many enzymes, particularly in one single family, with highly conserved structures and folds exhibit rather distinct substrate specificities. The underlying mechanism remains elusive, the resolution of which is of great importance for biochemistry, biophysics, and bioengineering. Here, we performed a neutron scattering experiment and molecular dynamics (MD) simulations on two structurally similar CYP450 proteins; CYP101 primarily catalyzes one type of ligands, then CYP2C9 can catalyze a large range of substrates. We demonstrated that it is the high density of salt bridges in CYP101 that reduces its structural flexibility, which controls the ligand access channel and the fluctuation of the catalytic pocket, thus restricting its selection on substrates. Moreover, we performed MD simulations on 146 different kinds of CYP450 proteins, spanning distinct biological categories including Fungi, Archaea, Bacteria, Protista, Animalia, and Plantae, and found the above mechanism generally valid. We demonstrated that, by fine changes of chemistry (salt-bridge density), the CYP450 superfamily can vary the structural flexibility of its member proteins among different biological categories, and thus differentiate their substrate specificities to meet the specific biological needs. As this mechanism is well-controllable and easy to be implemented, we expect it to be generally applicable in future enzymatic engineering to develop proteins of desired substrate specificities.


2021 ◽  
Vol 7 (9) ◽  
pp. 768
Author(s):  
Mario Aguiar ◽  
Thomas Orasch ◽  
Matthias Misslinger ◽  
Anna-Maria Dietl ◽  
Fabio Gsaller ◽  
...  

Siderophore-mediated acquisition of iron has been shown to be indispensable for the virulence of several fungal pathogens, the siderophore transporter Sit1 was found to mediate uptake of the novel antifungal drug VL-2397, and siderophores were shown to be useful as biomarkers as well as for imaging of fungal infections. However, siderophore uptake in filamentous fungi is poorly characterized. The opportunistic human pathogen Aspergillus fumigatus possesses five putative siderophore transporters. Here, we demonstrate that the siderophore transporters Sit1 and Sit2 have overlapping, as well as unique, substrate specificities. With respect to ferrichrome-type siderophores, the utilization of ferrirhodin and ferrirubin depended exclusively on Sit2, use of ferrichrome A depended mainly on Sit1, and utilization of ferrichrome, ferricrocin, and ferrichrysin was mediated by both transporters. Moreover, both Sit1 and Sit2 mediated use of the coprogen-type siderophores coprogen and coprogen B, while only Sit1 transported the bacterial ferrioxamine-type xenosiderophores ferrioxamines B, G, and E. Neither Sit1 nor Sit2 were important for the utilization of the endogenous siderophores fusarinine C and triacetylfusarinine C. Furthermore, A. fumigatus was found to lack utilization of the xenosiderophores schizokinen, basidiochrome, rhizoferrin, ornibactin, rhodotorulic acid, and enterobactin. Taken together, this study characterized siderophore use by A. fumigatus and substrate characteristics of Sit1 and Sit2.


2021 ◽  
Author(s):  
Jinxi Wang ◽  
Grace Ciampa ◽  
Dong Zheng ◽  
Qian Shi ◽  
Biyi Chen ◽  
...  

Calpain proteolysis contributes to the pathogenesis of heart failure but the calpain isoforms responsible and their substrate specificities have not been rigorously defined. One substrate, Junctophilin-2 (JP2), is essential for maintaining excitation-contraction coupling. We previously demonstrated that JP2 is cleaved by calpain-1 (CAPN1) between Arginine 565 (R565) and Threonine 566 (T566). Recently, calpain-2 (CAPN2) was reported to cleave JP2 at a novel site between Glycine 482 (G482) and Threonine 483 (T483). We aimed to directly compare the contributions of each calpain isoform, their Ca2+ sensitivity, and their cleavage site selection for JP2. We find CAPN1, CAPN2 and their requisite CAPNS1 regulatory subunit are induced by pressure overload stress that is concurrent with JP2 cleavage. Using in vitro calpain cleavage assays, we demonstrate that CAPN1 and CAPN2 cleave JP2 into similar 75-kD N-terminal (JP2NT) and 25-kD C-terminal fragments (JP2CT) with CAPNS1 co-expression enhancing proteolysis. Deletion mutagenesis shows both CAPN1 and CAPN2 require R565/T566 but not G482/T483. When heterologously expressed, the JP2CT peptide corresponding to R565/T566 cleavage approximates the 25-kD species found during cardiac stress while the C-terminal peptide from potential cleavage at G482/T483 produces a 35-kD product. Similar results were obtained for human JP2. Finally, we show that CAPN1 has higher Ca2+ sensitivity and cleavage efficacy than CAPN2 on JP2 and other cardiac substrates including cTnT, cTnI and β2-spectrin. We conclude that CAPN2 cleaves JP2 at the same functionally conserved R565/T566 site as CAPN1 but with less efficacy and suggest heart failure may be targeted through specific inhibition of CAPN1.


Author(s):  
Hiroya Tomita ◽  
Yohei Katsuyama ◽  
Yasuo Ohnishi

Abstract Nitroaromatic compounds are essential materials for chemical industry, but they are also potentially toxic environmental pollutants. Therefore, their sensitive detection and degradation are important concerns. The microbial degradation pathways of nitroaromatic compounds have been studied in detail, but their usefulness needs to be evaluated to understand their potential applications in bioremediation. Here, we developed a rapid and relatively sensitive assay system to evaluate the activities and substrate specificities of nitroaromatic dioxygenases involved in the oxidative biodegradation of nitroaromatic compounds. In this system, nitrous acid, which was released from the nitroaromatic compounds by the dioxygenases, was detected and quantified using the Saltzman reagent. Escherichia coli producing the 3-nitrobenzoic acid dioxygenase complex MnbAB from Comamonas sp. JS46 clearly showed the apparent substrate specificity of MnbAB as follows. MnbAB accepted not only 3-nitrobenzoic acid but also several other p- and m-nitrobenzoic acid derivatives as substrates, although it much preferred 3-nitrobenzoic acid to others. Furthermore, the presence of a hydroxy or an amino group at the ortho position of the nitro group decreased the activity of MnbAB. In addition, MnbAB accepted 2-(4-nitrophenyl)acetic acid as a substrate, which has one additional methylene group between the aromatic ring and the carboxy group of 3-nitrobenzoic acid. This is the first report about the detailed substrate specificity of MnbAB. Our system can be used for other nitroaromatic dioxygenases and contribute to their characterization.


2021 ◽  
Author(s):  
Pedro Dinis ◽  
Heli Tirkkonen ◽  
Vilja Siitonen ◽  
Benjamin Nji Wandi ◽  
Jarmo Niemi ◽  
...  

Streptomyces soil bacteria produce hundreds of anthracycline anticancer agents with a relatively conserved set of genes. This diversity depends on the rapid evolution of biosynthetic enzymes to acquire novel functionalities. Previous work has identified S-adenosyl-L-methionine -dependent methyltransferase-like proteins that catalyze either 4-O-methylation, 10-decarboxylation or 10-hydroxylation, with additional differences in substrate specificities. Here we focused on four protein regions to generate chimeric enzymes using sequences from four distinct subfamilies to elucidate their influence in catalysis. Combined with structural studies we managed to depict factors that influence gain-of-hydroxylation, loss-of-methylation and substrate selection. The engineering expanded the catalytic repertoire to include novel 9,10-elimination activity, and 4-O-methylation and 10-decarboxylation of unnatural substrates. The work provides an instructive account on how the rise of diversity of microbial natural products may occur through subtle changes in biosynthetic enzymes.


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