Probing molecular function of trypanosomal sialidases: single point mutations can change substrate specificity and increase hydrolytic activity
Sialidases are present on the surface of several trypanosomatid protozoan parasites. They are highly specific for sialic acid linked in alpha-(2,3) to a terminal beta-galactose and include the strictly hydrolytic enzymes and trans-sialidases (sialyl-transferases). Based on the structural comparison...
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| Veröffentlicht in: | Glycobiology (Oxford) 2001-04, Vol.11 (4), p.305-311 |
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| creator | Paris, G Cremona, M L Amaya, M F Buschiazzo, A Giambiagi, S Frasch, A C Alzari, P M |
| description | Sialidases are present on the surface of several trypanosomatid protozoan parasites. They are highly specific for sialic acid linked in alpha-(2,3) to a terminal beta-galactose and include the strictly hydrolytic enzymes and trans-sialidases (sialyl-transferases). Based on the structural comparison of the sialidase from Trypanosoma rangeli and the trans-sialidase from T. cruzi (the agent of Chagas' disease in humans), we have explored the role of specific amino acid residues sought to be important for substrate specificity. The substitution of a conserved tryptophanyl residue in the two enzymes, Trp312/313-Ala, changed substrate specificity, rendering the point mutants capable to hydrolyze both alpha-(2,3)- and alpha-(2,6)-linked sialoconjugates. The same mutation abolished sialyl-transferase activity, indicating that transfer (but not hydrolysis) requires a precise orientation of the bound substrate. The exchange substitution of another residue that modulates oligosaccharide binding, Gln284-Pro, was found to significantly increase the hydrolytic activity of sialidase, and residue Tyr119 was confirmed to be part of a second binding site for the acceptor substrate in trans-sialidase. Together with the structural information, these results provide a consistent framework to account for the unique enzymatic properties of trypanosome trans-sialidases. |
| doi_str_mv | 10.1093/glycob/11.4.305 |
| format | Article |
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They are highly specific for sialic acid linked in alpha-(2,3) to a terminal beta-galactose and include the strictly hydrolytic enzymes and trans-sialidases (sialyl-transferases). Based on the structural comparison of the sialidase from Trypanosoma rangeli and the trans-sialidase from T. cruzi (the agent of Chagas' disease in humans), we have explored the role of specific amino acid residues sought to be important for substrate specificity. The substitution of a conserved tryptophanyl residue in the two enzymes, Trp312/313-Ala, changed substrate specificity, rendering the point mutants capable to hydrolyze both alpha-(2,3)- and alpha-(2,6)-linked sialoconjugates. The same mutation abolished sialyl-transferase activity, indicating that transfer (but not hydrolysis) requires a precise orientation of the bound substrate. The exchange substitution of another residue that modulates oligosaccharide binding, Gln284-Pro, was found to significantly increase the hydrolytic activity of sialidase, and residue Tyr119 was confirmed to be part of a second binding site for the acceptor substrate in trans-sialidase. 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They are highly specific for sialic acid linked in alpha-(2,3) to a terminal beta-galactose and include the strictly hydrolytic enzymes and trans-sialidases (sialyl-transferases). Based on the structural comparison of the sialidase from Trypanosoma rangeli and the trans-sialidase from T. cruzi (the agent of Chagas' disease in humans), we have explored the role of specific amino acid residues sought to be important for substrate specificity. The substitution of a conserved tryptophanyl residue in the two enzymes, Trp312/313-Ala, changed substrate specificity, rendering the point mutants capable to hydrolyze both alpha-(2,3)- and alpha-(2,6)-linked sialoconjugates. The same mutation abolished sialyl-transferase activity, indicating that transfer (but not hydrolysis) requires a precise orientation of the bound substrate. The exchange substitution of another residue that modulates oligosaccharide binding, Gln284-Pro, was found to significantly increase the hydrolytic activity of sialidase, and residue Tyr119 was confirmed to be part of a second binding site for the acceptor substrate in trans-sialidase. Together with the structural information, these results provide a consistent framework to account for the unique enzymatic properties of trypanosome trans-sialidases.</description><subject>Amino Acid Substitution</subject><subject>Amino Acid Substitution - genetics</subject><subject>Amino Acids</subject><subject>Amino Acids - analysis</subject><subject>Animals</subject><subject>Binding Sites</subject><subject>Binding, Competitive</subject><subject>Biochemistry</subject><subject>Biochemistry, Molecular Biology</subject><subject>Chemical Sciences</subject><subject>Cristallography</subject><subject>Hydrolysis</subject><subject>Life Sciences</subject><subject>Microbiology and Parasitology</subject><subject>Models, Molecular</subject><subject>Mutagenesis, Site-Directed</subject><subject>Neuraminidase</subject><subject>Neuraminidase - chemistry</subject><subject>Neuraminidase - genetics</subject><subject>Neuraminidase - metabolism</subject><subject>Parasitology</subject><subject>Point Mutation</subject><subject>Point Mutation - genetics</subject><subject>Protein Conformation</subject><subject>Substrate Specificity</subject><subject>Trypanosoma</subject><subject>Trypanosoma - enzymology</subject><subject>Trypanosoma - genetics</subject><subject>Trypanosoma rangeli</subject><issn>0959-6658</issn><issn>1460-2423</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpdkUFv1DAQhS0EotvCmRuyOHDLrsd2nIRbVQFFWgkOcLYcr73ryrGD7VTKr-Av42pXVOI07_C9NzN6CL0DsgUysN3RrzqOO4At3zLSvkAb4II0lFP2Em3I0A6NEG1_ha5zfiAEBPTta3QFwNq-74YN-vMjxdGFI56iN3rxKmG7BF1cDDhaXNI6qxBznJTH2SnvDiqb_KnqcPQGz9GFgqelqCdHxloFrE8qHA3Oy5hLUqWq2WhnnXZlxSocsAs6mRqDT-shRb8Wp7GqKx8r8Aa9sspn8_Yyb9CvL59_3t03--9fv93d7hvNGSmNBT0qCrSDlvS9HawWipGxG1rB9UiIOFjoNXQdpRQM59ZQargiFHpOR96yG9Scc0_Kyzm5SaVVRuXk_e1ezioXsyRJaNtyMpBHqPzHMz-n-HsxucjJZW28V8HEJUtKOOs7Riv44T_wIS4p1F8kBcKIYEJUaHeGdIo5J2P_nQBEPhUrz8VKAMllLbY63l9il3Eyh2f-0iT7CxUyoo4</recordid><startdate>20010401</startdate><enddate>20010401</enddate><creator>Paris, G</creator><creator>Cremona, M L</creator><creator>Amaya, M F</creator><creator>Buschiazzo, A</creator><creator>Giambiagi, S</creator><creator>Frasch, A C</creator><creator>Alzari, P M</creator><general>Oxford Publishing Limited (England)</general><general>Oxford University Press (OUP)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QO</scope><scope>7TK</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>1XC</scope><orcidid>https://orcid.org/0000-0002-2509-6526</orcidid></search><sort><creationdate>20010401</creationdate><title>Probing molecular function of trypanosomal sialidases: single point mutations can change substrate specificity and increase hydrolytic activity</title><author>Paris, G ; Cremona, M L ; Amaya, M F ; Buschiazzo, A ; Giambiagi, S ; Frasch, A C ; Alzari, P M</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c430t-f1cba212715088f9fc6a30b79564cb006df18c1772221e44fe22e4a021842b453</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Amino Acid Substitution</topic><topic>Amino Acid Substitution - genetics</topic><topic>Amino Acids</topic><topic>Amino Acids - analysis</topic><topic>Animals</topic><topic>Binding Sites</topic><topic>Binding, Competitive</topic><topic>Biochemistry</topic><topic>Biochemistry, Molecular Biology</topic><topic>Chemical Sciences</topic><topic>Cristallography</topic><topic>Hydrolysis</topic><topic>Life Sciences</topic><topic>Microbiology and Parasitology</topic><topic>Models, Molecular</topic><topic>Mutagenesis, Site-Directed</topic><topic>Neuraminidase</topic><topic>Neuraminidase - chemistry</topic><topic>Neuraminidase - genetics</topic><topic>Neuraminidase - metabolism</topic><topic>Parasitology</topic><topic>Point Mutation</topic><topic>Point Mutation - genetics</topic><topic>Protein Conformation</topic><topic>Substrate Specificity</topic><topic>Trypanosoma</topic><topic>Trypanosoma - enzymology</topic><topic>Trypanosoma - genetics</topic><topic>Trypanosoma rangeli</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Paris, G</creatorcontrib><creatorcontrib>Cremona, M L</creatorcontrib><creatorcontrib>Amaya, M F</creatorcontrib><creatorcontrib>Buschiazzo, A</creatorcontrib><creatorcontrib>Giambiagi, S</creatorcontrib><creatorcontrib>Frasch, A C</creatorcontrib><creatorcontrib>Alzari, P M</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>Hyper Article en Ligne (HAL)</collection><jtitle>Glycobiology (Oxford)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Paris, G</au><au>Cremona, M L</au><au>Amaya, M F</au><au>Buschiazzo, A</au><au>Giambiagi, S</au><au>Frasch, A C</au><au>Alzari, P M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Probing molecular function of trypanosomal sialidases: single point mutations can change substrate specificity and increase hydrolytic activity</atitle><jtitle>Glycobiology (Oxford)</jtitle><addtitle>Glycobiology</addtitle><date>2001-04-01</date><risdate>2001</risdate><volume>11</volume><issue>4</issue><spage>305</spage><epage>311</epage><pages>305-311</pages><issn>0959-6658</issn><eissn>1460-2423</eissn><abstract>Sialidases are present on the surface of several trypanosomatid protozoan parasites. 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The exchange substitution of another residue that modulates oligosaccharide binding, Gln284-Pro, was found to significantly increase the hydrolytic activity of sialidase, and residue Tyr119 was confirmed to be part of a second binding site for the acceptor substrate in trans-sialidase. Together with the structural information, these results provide a consistent framework to account for the unique enzymatic properties of trypanosome trans-sialidases.</abstract><cop>England</cop><pub>Oxford Publishing Limited (England)</pub><pmid>11358879</pmid><doi>10.1093/glycob/11.4.305</doi><tpages>7</tpages><orcidid>https://orcid.org/0000-0002-2509-6526</orcidid><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Oxford University Press Journals All Titles (1996-Current); Alma/SFX Local Collection |
| subjects | Amino Acid Substitution Amino Acid Substitution - genetics Amino Acids Amino Acids - analysis Animals Binding Sites Binding, Competitive Biochemistry Biochemistry, Molecular Biology Chemical Sciences Cristallography Hydrolysis Life Sciences Microbiology and Parasitology Models, Molecular Mutagenesis, Site-Directed Neuraminidase Neuraminidase - chemistry Neuraminidase - genetics Neuraminidase - metabolism Parasitology Point Mutation Point Mutation - genetics Protein Conformation Substrate Specificity Trypanosoma Trypanosoma - enzymology Trypanosoma - genetics Trypanosoma rangeli |
| title | Probing molecular function of trypanosomal sialidases: single point mutations can change substrate specificity and increase hydrolytic activity |
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