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3nra

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    {{ template.Protein{ leadContact:"", title:"Crystal structure of an aspartate aminotransferase (YP_354942.1) from Rhodobacter sphaeroides 2.4.1 at 2.15 A resolution. To be published",site:'JCSG', status:'In PDB', date:"2010-06-30", targetid:"403422", pdbid:"3nra", source:"Rhodobacter sphaeroides 2.4.1", relatedPDBs:[], refids:"YP_354942.1, 3.40.640.10, 332507", molwt:"43542.97", residues:"406", isopoint:"5.89", sequence:"msieakfkklgtdnapgqevrqsaaglealirgapiegrpvdfshgdvdaheptpgafdlfsagvqsgg vqayteyrgdlgirdllaprlaaftgapvdardgliitpgtqgalflavaatvargdkvaivqpdyfan rklveffegemvpvqldyvsadetragldltgleeafkagarvflfsnpnnpagvvysaeeigqiaala arygatviadqlysrlryagasythlraeaavdaenvvtimgpskteslsgyrlgvafgsraiiarmek lqaivslraagysqavlrgwfdeapgwmedriarhqairdellhvlrgcegvfartpqagsylfprlpk lavapaefvkilrlqagvvvtpgtefsphtadsvrlnfsqdheaavaaarrivtlveryra", method:"XRAY", numchains:"2", resolution:"2.15", rfree:"0.200", mattcoeff:"2.40", rfactor:"0.153", waters:"622", solcontent:"48.76", ligands:"", metals:"", model:"False", uniprot:"Q3IWP3", pubmed:"" } }}

    ...

    ************************************************************************************************************************************************

    Kinetic analysis of the aspartate aminotransferase 3NRA from Rhodobacter sphaeroides

    ...

    The structure of the protein encoded by gene RSP_3439 from Rhodobacter sphaeroides (YP_354942.1 ), was determined to 2.15 Å by the JCSG(PDB id 3NRA). The protein was identified as a putative aspartate aminotransferase (RsAAT; 2.6.1.1) based on structural similarity to other aspartate aminotransferases.

    ...

    Sequence analysis of RsAAT indicates that it is a member of fold-type I of PLP-dependent enzymes, specifically subfamily I (PF00155) a conserved family that includes aspartate, tyrosine, alanine, phenylalanine and histidinol-phosphate aminotransferases.1 Structural comparison of RsAAT using the DALI server revealed structural similarities with PLP-dependent aspartate aminotransferases and a PLP-dependent aromatic aminotransferase, 1DJU from Pyrococcus horikoshii (PhAAT)2,3. These sequence and structural comparisons indicate that RsAAT belongs to the alpha-division of subfamily I, whose prokaryotic members show broad specificity that includes aromatic amino acids4.

    ...

    Aspartate aminotransferases reversibly catalyze the transamination reaction between L-aspartate and 2-oxoglutarate to yield oxaloacetate and L-glutamate through a ping-pong bi-bi mechanism mediated by the coenzyme PLP5-8. In RsAAT, PLP is covalently bound to the Lys252 residue situated in the active site cleft, which is highly homologous across the (PLP)-dependent aspartate aminotransferase superfamily (fold-type I)9.  The other residues present at the active site are Gly109, Thr110, Gln111, Tyr135, Asn189, Asp217, Tyr220, Gly249, Ser251 Arg260, Tyr338, and Tyr7310. These active site residues are conserved amongst a number of similar proteins: 1GDE, 1J32, 1DJU, 1BJW, and 1ASL (PDB ids).

    ...

    Kinetics data was obtained spectrophotometrically using malate dehydrogenase (MDH) as a coupling enzyme. Malate dehydrogenase converts the oxaloacetate, produced by the RsAAT, to malate with the concomitant oxidation of NADH to NAD+. The reaction is monitored at 340 nm, where NADH absorbs strongly but NAD+ does not. Reaction mixtures consisted of 100 mM Tris or 20 mM KH2PO4 (both pH 8.0), 0.2 mM NADH, 10 mM 2-oxoglutarate, 15 μM PLP, 3.6 U mL-1 porcine heart MDH, and 2 µM 3NRA protein. L-aspartate, was varied from 0 to 20 mM.

    ...

    While a protein concentration dependent reaction rate was observed for the transamination reaction in both phosphate and Tris buffers, enzymatic activity was significantly lower in 20 mM KH2PO4 pH 8.0 when compared to rates in  100 mM Tris pH 8.0 (Figure 1). This is in agreement with published reports that phosphate can interfere with activity11. RsAAT activity follows Michaelis-Menten kinetics with respect to L-aspartate: Km of 2.1 mM and a kcat of 0.016 s-1 (Figure 2). While the Km for RsAAT is in agreement with other aspartate aminotransferase homologues with reported kinetic parameters (5 mM for 1J32; 2.0 mM for 1BJW, 1.9 mM for 1ASL), the kcat for RsAAT is orders of magnitude lower (187 s-1 for 1J32 and 259 s-1 for 1ASL).

    ...

    BioLEd Contributors: Nikolas Hayes, Anita Or, Payal Patel, Michael Pokrass, Paul Riehl, Victor Teran, Joseph Tilitsky, Jennifer Tomlinson Kaitlin Bailey, Ellen Schleckman, Cameron Mura, Carol Price, Linda Columbus. Funded by NSF DUE 1044858.

    ...

    References

    1. Finn R. D., Mistry J., Tate J., Coggill P., Heger A., Pollington J. E., Gavin, O. L., Gunasekaran, P., Ceric, G., Forslund, K., Holm, L., Sonnhammer, E. L. L., Eddy, S. R., Bateman, A. 2010. The Pfam Protein Families Database. Nucleic Acids Research. 38:211-222.

    2. Holm L., Rosenstrom P. 2010. Dali Server: Conservation Mapping in 3D. Nucleic Acids Research. 38:545-549.

    3. Matsui I, Matsui E, Sakai Y, Kikuchi H, Kawarabayasi Y, Ura H, Kawaguchi S, Kuramitsu S, Harata K. 2000. The molecular structure of hyperthermostable aromatic aminotransferase with novel substrate specificity from Pyrococcus horikoshii. J. Biol. Chem. 275:4871–4879.

    4. Jensen, R., Gu, W. 1996. Evolutionary Recruitment of Biochemically Specialized Subdivisions of Family I within Protein Superfamily of Aminotransferases. J. Bacteriology.178(8):2161-2171.

    5. Kameya, M., Arai, H., Ishii, M., and Igarashi, Y. 2010. Purification of three aminotransferases from Hydogenobacter thermophilus TK-6-novel type of alanine or glycine aminotransferase. FEBS Journal.277(8): 1876-85. 

    6. Hayashi, H., Mizuguchi, H., Miyahara, I., Nakajima, Y., Hirotsu, K., Kagmiyama, H. 2003. Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis. J.Biol.Chem. 278:9481-9488.

    7. de la Torre, F., De Santis, L., Suarez, M. F., Crespillo, R., Canovas, F. M. 2006. Identification and functional analysis of a prokaryotic-type aspartate aminotransferase: implications for plant amino acid metabolism. Plant Journal. 46:414-425.

    8. Wu, H., Yang, Y., Wang, S., Qiao, J., Xia, Y., Wang, Y., Gao, S. F., Liu, J., Xue, P. Q., Gao, X. W. 2011. Cloning, expression and characterization of a new aspartate aminotransferase from Bacillus subtilis B3. Febs Journal. 278:1345-1357.

    9. Wrenger, C., Mueller, I. B., Schifferdecker, A. J., Jain, R., Jordanova, R., Groves, M. R. 2011 Specific Inhibition of the Aspartate Aminotransferase of Plasmodium falciparum. J.Mol.Biol.;405:956-971.

    10. Yano, T., Kuramitsu, S., Tanase, S., Morino, Y., and Kagamiyama, H. 1992. Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase. Biochemistry. 31: 5878-5887.

    11. Rej, R., Vanderlinde, R. E. 1975. Effects of buffers on aspartate-aminotransferase activity and association of enzyme with pyridoxal-phosphate. Clin Chem. 21(11):1585-1591.

    ...


    Other changes:

    1. /body/p[3]/a[4]/@class: " external""external"
    2. /body/p[6]/a/@class: " external""external"

    Version from 01:55, 9 May 2012

    This revision modified by Admin (Ban)
    {{ template.Protein{ leadContact:"", title:"Crystal structure of an aspartate aminotransferase (YP_354942.1) from Rhodobacter sphaeroides 2.4.1 at 2.15 A resolution. To be published",site:'JCSG', status:'In PDB', date:"2010-06-30", targetid:"403422", pdbid:"3nra", source:"Rhodobacter sphaeroides 2.4.1", relatedPDBs:[], refids:"YP_354942.1, 3.40.640.10, 332507", molwt:"43542.97", residues:"406", isopoint:"5.89", sequence:"msieakfkklgtdnapgqevrqsaaglealirgapiegrpvdfshgdvdaheptpgafdlfsagvqsgg vqayteyrgdlgirdllaprlaaftgapvdardgliitpgtqgalflavaatvargdkvaivqpdyfan rklveffegemvpvqldyvsadetragldltgleeafkagarvflfsnpnnpagvvysaeeigqiaala arygatviadqlysrlryagasythlraeaavdaenvvtimgpskteslsgyrlgvafgsraiiarmek lqaivslraagysqavlrgwfdeapgwmedriarhqairdellhvlrgcegvfartpqagsylfprlpk lavapaefvkilrlqagvvvtpgtefsphtadsvrlnfsqdheaavaaarrivtlveryra", method:"XRAY", numchains:"2", resolution:"2.15", rfree:"0.200", mattcoeff:"2.40", rfactor:"0.153", waters:"622", solcontent:"48.76", ligands:"", metals:"", model:"False", uniprot:"Q3IWP3", pubmed:"" } }}

    ...


    Current version

    This revision modified by cawprice (Ban)
    {{ template.Protein{ leadContact:"", title:"Crystal structure of an aspartate aminotransferase (YP_354942.1) from Rhodobacter sphaeroides 2.4.1 at 2.15 A resolution. To be published",site:'JCSG', status:'In PDB', date:"2010-06-30", targetid:"403422", pdbid:"3nra", source:"Rhodobacter sphaeroides 2.4.1", relatedPDBs:[], refids:"YP_354942.1, 3.40.640.10, 332507", molwt:"43542.97", residues:"406", isopoint:"5.89", sequence:"msieakfkklgtdnapgqevrqsaaglealirgapiegrpvdfshgdvdaheptpgafdlfsagvqsgg vqayteyrgdlgirdllaprlaaftgapvdardgliitpgtqgalflavaatvargdkvaivqpdyfan rklveffegemvpvqldyvsadetragldltgleeafkagarvflfsnpnnpagvvysaeeigqiaala arygatviadqlysrlryagasythlraeaavdaenvvtimgpskteslsgyrlgvafgsraiiarmek lqaivslraagysqavlrgwfdeapgwmedriarhqairdellhvlrgcegvfartpqagsylfprlpk lavapaefvkilrlqagvvvtpgtefsphtadsvrlnfsqdheaavaaarrivtlveryra", method:"XRAY", numchains:"2", resolution:"2.15", rfree:"0.200", mattcoeff:"2.40", rfactor:"0.153", waters:"622", solcontent:"48.76", ligands:"", metals:"", model:"False", uniprot:"Q3IWP3", pubmed:"" } }}

    ...

    ************************************************************************************************************************************************

    Kinetic analysis of the aspartate aminotransferase 3NRA from Rhodobacter sphaeroides

    ...

    The structure of the protein encoded by gene RSP_3439 from Rhodobacter sphaeroides (YP_354942.1 ), was determined to 2.15 Å by the JCSG(PDB id 3NRA). The protein was identified as a putative aspartate aminotransferase (RsAAT; 2.6.1.1) based on structural similarity to other aspartate aminotransferases.

    ...

    Sequence analysis of RsAAT indicates that it is a member of fold-type I of PLP-dependent enzymes, specifically subfamily I (PF00155) a conserved family that includes aspartate, tyrosine, alanine, phenylalanine and histidinol-phosphate aminotransferases.1 Structural comparison of RsAAT using the DALI server revealed structural similarities with PLP-dependent aspartate aminotransferases and a PLP-dependent aromatic aminotransferase, 1DJU from Pyrococcus horikoshii (PhAAT)2,3. These sequence and structural comparisons indicate that RsAAT belongs to the alpha-division of subfamily I, whose prokaryotic members show broad specificity that includes aromatic amino acids4.

    ...

    Aspartate aminotransferases reversibly catalyze the transamination reaction between L-aspartate and 2-oxoglutarate to yield oxaloacetate and L-glutamate through a ping-pong bi-bi mechanism mediated by the coenzyme PLP5-8. In RsAAT, PLP is covalently bound to the Lys252 residue situated in the active site cleft, which is highly homologous across the (PLP)-dependent aspartate aminotransferase superfamily (fold-type I)9.  The other residues present at the active site are Gly109, Thr110, Gln111, Tyr135, Asn189, Asp217, Tyr220, Gly249, Ser251 Arg260, Tyr338, and Tyr7310. These active site residues are conserved amongst a number of similar proteins: 1GDE, 1J32, 1DJU, 1BJW, and 1ASL (PDB ids).

    ...

    Kinetics data was obtained spectrophotometrically using malate dehydrogenase (MDH) as a coupling enzyme. Malate dehydrogenase converts the oxaloacetate, produced by the RsAAT, to malate with the concomitant oxidation of NADH to NAD+. The reaction is monitored at 340 nm, where NADH absorbs strongly but NAD+ does not. Reaction mixtures consisted of 100 mM Tris or 20 mM KH2PO4 (both pH 8.0), 0.2 mM NADH, 10 mM 2-oxoglutarate, 15 μM PLP, 3.6 U mL-1 porcine heart MDH, and 2 µM 3NRA protein. L-aspartate, was varied from 0 to 20 mM.

    ...

    While a protein concentration dependent reaction rate was observed for the transamination reaction in both phosphate and Tris buffers, enzymatic activity was significantly lower in 20 mM KH2PO4 pH 8.0 when compared to rates in  100 mM Tris pH 8.0 (Figure 1). This is in agreement with published reports that phosphate can interfere with activity11. RsAAT activity follows Michaelis-Menten kinetics with respect to L-aspartate: Km of 2.1 mM and a kcat of 0.016 s-1 (Figure 2). While the Km for RsAAT is in agreement with other aspartate aminotransferase homologues with reported kinetic parameters (5 mM for 1J32; 2.0 mM for 1BJW, 1.9 mM for 1ASL), the kcat for RsAAT is orders of magnitude lower (187 s-1 for 1J32 and 259 s-1 for 1ASL).

    ...

    BioLEd Contributors: Nikolas Hayes, Anita Or, Payal Patel, Michael Pokrass, Paul Riehl, Victor Teran, Joseph Tilitsky, Jennifer Tomlinson Kaitlin Bailey, Ellen Schleckman, Cameron Mura, Carol Price, Linda Columbus. Funded by NSF DUE 1044858.

    ...

    References

    1. Finn R. D., Mistry J., Tate J., Coggill P., Heger A., Pollington J. E., Gavin, O. L., Gunasekaran, P., Ceric, G., Forslund, K., Holm, L., Sonnhammer, E. L. L., Eddy, S. R., Bateman, A. 2010. The Pfam Protein Families Database. Nucleic Acids Research. 38:211-222.

    2. Holm L., Rosenstrom P. 2010. Dali Server: Conservation Mapping in 3D. Nucleic Acids Research. 38:545-549.

    3. Matsui I, Matsui E, Sakai Y, Kikuchi H, Kawarabayasi Y, Ura H, Kawaguchi S, Kuramitsu S, Harata K. 2000. The molecular structure of hyperthermostable aromatic aminotransferase with novel substrate specificity from Pyrococcus horikoshii. J. Biol. Chem. 275:4871–4879.

    4. Jensen, R., Gu, W. 1996. Evolutionary Recruitment of Biochemically Specialized Subdivisions of Family I within Protein Superfamily of Aminotransferases. J. Bacteriology.178(8):2161-2171.

    5. Kameya, M., Arai, H., Ishii, M., and Igarashi, Y. 2010. Purification of three aminotransferases from Hydogenobacter thermophilus TK-6-novel type of alanine or glycine aminotransferase. FEBS Journal.277(8): 1876-85. 

    6. Hayashi, H., Mizuguchi, H., Miyahara, I., Nakajima, Y., Hirotsu, K., Kagmiyama, H. 2003. Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis. J.Biol.Chem. 278:9481-9488.

    7. de la Torre, F., De Santis, L., Suarez, M. F., Crespillo, R., Canovas, F. M. 2006. Identification and functional analysis of a prokaryotic-type aspartate aminotransferase: implications for plant amino acid metabolism. Plant Journal. 46:414-425.

    8. Wu, H., Yang, Y., Wang, S., Qiao, J., Xia, Y., Wang, Y., Gao, S. F., Liu, J., Xue, P. Q., Gao, X. W. 2011. Cloning, expression and characterization of a new aspartate aminotransferase from Bacillus subtilis B3. Febs Journal. 278:1345-1357.

    9. Wrenger, C., Mueller, I. B., Schifferdecker, A. J., Jain, R., Jordanova, R., Groves, M. R. 2011 Specific Inhibition of the Aspartate Aminotransferase of Plasmodium falciparum. J.Mol.Biol.;405:956-971.

    10. Yano, T., Kuramitsu, S., Tanase, S., Morino, Y., and Kagamiyama, H. 1992. Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase. Biochemistry. 31: 5878-5887.

    11. Rej, R., Vanderlinde, R. E. 1975. Effects of buffers on aspartate-aminotransferase activity and association of enzyme with pyridoxal-phosphate. Clin Chem. 21(11):1585-1591.

    ...


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