scholarly journals Substrate Specificity of Chimeric Enzymes Formed by Interchange of the Catalytic and Specificity Domains of the 5′-Nucleotidase UshA and the 3′-Nucleotidase CpdB

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2307
Author(s):  
Alicia Cabezas ◽  
Iralis López-Villamizar ◽  
María Jesús Costas ◽  
José Carlos Cameselle ◽  
João Meireles Ribeiro
Keyword(s):  
Catalytic Domain ◽  
Nucleotidase Activity ◽  
Catalytic Behavior ◽  
Substrate Specificities ◽  
Chimeric Enzymes ◽  
Domain Structures ◽  
The Common ◽  
Partial Overlap ◽  
Specificity Domain ◽  
Terminal Domains

The 5′-nucleotidase UshA and the 3′-nucleotidase CpdB from Escherichia coli are broad-specificity phosphohydrolases with similar two-domain structures. Their N-terminal domains (UshA_Ndom and CpdB_Ndom) contain the catalytic site, and their C-terminal domains (UshA_Cdom and CpdB_Cdom) contain a substrate-binding site responsible for specificity. Both enzymes show only partial overlap in their substrate specificities. So, it was decided to investigate the catalytic behavior of chimeras bearing the UshA catalytic domain and the CpdB specificity domain, or vice versa. UshA_Ndom–CpdB_Cdom and CpdB_Ndom–UshA_Cdom were constructed and tested on substrates specific to UshA (5′-AMP, CDP-choline, UDP-glucose) or to CpdB (3′-AMP), as well as on 2′,3′-cAMP and on the common phosphodiester substrate bis-4-NPP (bis-4-nitrophenylphosphate). The chimeras did show neither 5′-nucleotidase nor 3′-nucleotidase activity. When compared to UshA, UshA_Ndom–CpdB_Cdom conserved high activity on bis-4-NPP, some on CDP-choline and UDP-glucose, and displayed activity on 2′,3′-cAMP. When compared to CpdB, CpdB_Ndom–UshA_Cdom conserved phosphodiesterase activities on 2′,3′-cAMP and bis-4-NPP, and gained activity on the phosphoanhydride CDP-choline. Therefore, the non-nucleotidase activities of UshA and CpdB are not fully dependent on the interplay between domains. The specificity domains may confer the chimeras some of the phosphodiester or phosphoanhydride selectivity displayed when associated with their native partners. Contrarily, the nucleotidase activity of UshA and CpdB depends strictly on the interplay between their native catalytic and specificity domains.

scholarly journals Plant β-tubulin phosphorylation on Ser172 as canonical suppressing factor of microtubule growth

2019 ◽  
Vol 24 ◽  
pp. 321-326
Author(s):  
P. A. Karpov ◽  
Ya. B. Blume
Keyword(s):  
Protein Kinases ◽  
Catalytic Domain ◽  
Homo Sapiens ◽  
Fruit Fly ◽  
Microtubule Assembly ◽  
Multiple Sequence ◽  
Significant Similarity ◽  
Domain Structures ◽  
Phylogenetic Profiling ◽  
Β Tubulin

Aim. The estimation of potential role of plant β-tubulin Ser172 phosphorylation for correct function of microtubules and cell division due to selection of protein kinases, most probable associated with phosphorylation of Ser172 in Arabidopsis thaliana (L.) Heynh. Methods. Literature and database search. Comparison of protein sequences and structures: multiple sequence alignment, phylogenetic profiling, protein structure modeling, etc. Results. Comparison of Ser172 site region from all known β-tubulins from Homo sapiens, Sus scrofa, Saccharomyces cerevisiae, Drosophila melanogaster and A. thaliana confirms its significant similarity. Joint clusterization of all Ser172 site regions (in S±10 a.a. format) reveals that plant site is most similar to Ser172±10 fragment of β-tubulin from S. cerevisiae. At the same time, sequences and catalytic domain structures of cyclin-dependent kinases 1 and YAK1-related kinases (MNB/DYRK1a/YAK1) associated with Ser172 phosphorylation, found maximal similarity in A. thaliana and S. cerevisiae. Сonclusions. The results confirm similarity of amino acid environment of Ser172 in β-tubulin isotypes in human, pig, fruit fly, yeast and arabidopsis. This suggests similar effect of β-tu­bulin phosphorylation at Ser172 for inhibition of microtubule assembly onto their protofilaments and its association with CDK1 and YAK1-related protein kinases. Similarity of Ser172 sites and associated protein kinases, allows us to expect similar effect of this modification on structure of microtubules in A. thaliana and S. cerevisiae. Keywords: β-tubulin, Ser172, phospho­rylation, CDK1, DYRK1, MNB, YAK1.  

scholarly journals Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Qingping Xu ◽  
Dominique Mengin-Lecreulx ◽  
Xueqian W. Liu ◽  
Delphine Patin ◽  
Carol L. Farr ◽  
...  
Keyword(s):  
Cell Wall ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Catalytic Domain ◽  
Substrate Binding ◽  
Diaminopimelic Acid ◽  
Single Mutation ◽  
Molecular Architecture ◽  
Substrate Specificities ◽  
Cell Wall Hydrolases

ABSTRACTBacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.

scholarly journals Identification of GH15 Family Thermophilic Archaeal Trehalases That Function within a Narrow Acidic-pH Range

2015 ◽  
Vol 81 (15) ◽  
pp. 4920-4931 ◽  
Author(s):  
Masayoshi Sakaguchi ◽  
Satoru Shimodaira ◽  
Shin-nosuke Ishida ◽  
Miko Amemiya ◽  
Shotaro Honda ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Glycoside Hydrolase Family ◽  
Acidic Ph ◽  
Content Type ◽  
Domain Structures ◽  
Cazy Database ◽  
Ph Range ◽  
And Function ◽  
Reduced Activity ◽  
Insight Into

ABSTRACTTwo glucoamylase-like genes,TVN1315andTa0286, from the archaeaThermoplasma volcaniumandT. acidophilum, respectively, were expressed inEscherichia coli. The gene products, TVN1315 and Ta0286, were identified as archaeal trehalases. These trehalases belong to the CAZy database family GH15, although they have putative (α/α)6barrel catalytic domain structures similar to those of GH37 and GH65 family trehalases from other organisms. These newly identified trehalases function within a narrow range of acidic pH values (pH 3.2 to 4.0) and at high temperatures (50 to 60°C), and these enzymes displayKmvalues for trehalose higher than those observed for typical trehalases. These enzymes were inhibited by validamycin A; however, the inhibition constants (Ki) were higher than those of other trehalases. Three TVN1315 mutants, corresponding to E408Q, E571Q, and E408Q/E571Q mutations, showed reduced activity, suggesting that these two glutamic acid residues are involved in trehalase catalysis in a manner similar to that of glucoamylase. To date, TVN1315 and Ta0286 are the first archaeal trehalases to be identified, and this is the first report of the heterologous expression of GH15 family trehalases. The identification of these trehalases could extend our understanding of the relationships between the structure and function of GH15 family enzymes as well as glycoside hydrolase family enzymes; additionally, these enzymes provide insight into archaeal trehalose metabolism.

scholarly journals Evolution-Inspired Engineering of Anthracycline Methyltransferases

2021 ◽  
Author(s):  
Pedro Dinis ◽  
Heli Tirkkonen ◽  
Vilja Siitonen ◽  
Benjamin Nji Wandi ◽  
Jarmo Niemi ◽  
...  
Keyword(s):  
Natural Products ◽  
Anticancer Agents ◽  
Soil Bacteria ◽  
Rapid Evolution ◽  
Biosynthetic Enzymes ◽  
Structural Studies ◽  
Substrate Selection ◽  
Substrate Specificities ◽  
Chimeric Enzymes ◽  
Microbial Natural Products

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.

scholarly journals The role of the Src homology domains in morphological transformation by v-src.

1997 ◽  
Vol 8 (7) ◽  
pp. 1183-1193 ◽  
Author(s):  
M Tian ◽  
G S Martin
Keyword(s):  
Tyrosine Phosphorylation ◽  
Catalytic Domain ◽  
Wild Type ◽  
Morphological Transformation ◽  
Sh2 Domains ◽  
C Terminus ◽  
Src Homology ◽  
Type V ◽  
Terminal Domains

The Src homology (SH2 and SH3) domains of v-Src are required for transformation of Rat-2 cells and for wild-type (morphr) transformation of chicken embryo fibroblasts (CEFs). We report herein that the N-terminal domains of v-Src, when expressed in trans, cannot complement the transformation defect of a deletion mutant lacking the "unique," SH3, and SH2 regions. However, the same regions of Src can promote transformation when translocated to the C terminus of v-Src, although the transformation of CEFs is somewhat slower. We conclude that the SH3 and SH2 domains must be present in cis to the catalytic domain to promote transformation but that transformation is not dependent on the precise intramolecular location of these domains. In CEFSs and in Rat-2 cells, the expression of wild-type v-Src results in tyrosine phosphorylation of proteins that bind to the v-Src SH3 and SH2 domains in vitro; mutations in the SH2 or SH3 and SH2 domains prevent the phosphorylation of these proteins. These findings are most consistent with models in which the SH3 and SH2 domains of v-Src directly or indirectly target the catalytic domain to substrates involved in transformation. However, the N-terminal domains of v-Src can promote tyrosine phosphorylation of certain proteins, in particular p130Cas, even when expressed in the absence of the catalytic domain, indicating that the N-terminal domains of v-Src have effects that are independent of the catalytic domain.

scholarly journals Substrate-specifying determinants of the nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2

2004 ◽  
Vol 381 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Anisoara CIMPEAN ◽  
Cristiana STEFAN ◽  
Rik GIJSBERS ◽  
Willy STALMANS ◽  
Mathieu BOLLEN
Keyword(s):  
Substrate Specificity ◽  
Metal Binding ◽  
Catalytic Domain ◽  
Polypeptide Chain ◽  
Catalytic Site ◽  
Domain Swapping ◽  
Lysophospholipase D ◽  
Terminal Domains

The nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2/autotaxin are structurally related eukaryotic ecto-enzymes, but display a very different substrate specificity. NPP1 releases nucleoside 5′-monophosphates from various nucleotides, whereas NPP2 mainly functions as a lysophospholipase D. We have used a domain-swapping approach to map substrate-specifying determinants of NPP1 and NPP2. The catalytic domain of NPP1 fused to the N- and C-terminal domains of NPP2 was hyperactive as a nucleotide phosphodiesterase, but did not show any lysophospholipase D activity. In contrast, chimaeras of the catalytic domain of NPP2 and the N- and/or C-terminal domains of NPP1 were completely inactive. These data indicate that the catalytic domain as well as both extremities of NPP2 contain lysophospholipid-specifying sequences. Within the catalytic domain of NPP1 and NPP2, we have mapped residues close to the catalytic site that determine the activities towards nucleotides and lysophospholipids. We also show that the conserved Gly/Phe-Xaa-Gly-Xaa-Xaa-Gly (G/FXGXXG) motif near the catalytic site is required for metal binding, but is not involved in substrate-specification. Our data suggest that the distinct activities of NPP1 and NPP2 stem from multiple differences throughout the polypeptide chain.

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