1nff
From Proteopedia
(New page: 200px<br /><applet load="1nff" size="450" color="white" frame="true" align="right" spinBox="true" caption="1nff, resolution 1.80Å" /> '''Crystal structure of...) |
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| - | [[Image:1nff.gif|left|200px]]<br /><applet load="1nff" size=" | + | [[Image:1nff.gif|left|200px]]<br /><applet load="1nff" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1nff, resolution 1.80Å" /> | caption="1nff, resolution 1.80Å" /> | ||
'''Crystal structure of Rv2002 gene product from Mycobacterium tuberculosis'''<br /> | '''Crystal structure of Rv2002 gene product from Mycobacterium tuberculosis'''<br /> | ||
==Overview== | ==Overview== | ||
| - | One of the serious bottlenecks in structural genomics projects is | + | One of the serious bottlenecks in structural genomics projects is overexpression of the target proteins in soluble form. We have applied the directed evolution technique and prepared soluble mutants of the Mycobacterium tuberculosis Rv2002 gene product, the wild type of which had been expressed as inclusion bodies in Escherichia coli. A triple mutant I6TV47MT69K (Rv2002-M3) was chosen for structural and functional characterizations. Enzymatic assays indicate that the Rv2002-M3 protein has a high catalytic activity as a NADH-dependent 3alpha, 20beta-hydroxysteroid dehydrogenase. We have determined the crystal structures of a binary complex with NAD(+) and a ternary complex with androsterone and NADH. The structure reveals that Asp-38 determines the cofactor specificity. The catalytic site includes the triad Ser-140Tyr-153Lys-157. Additionally, it has an unusual feature, Glu-142. Enzymatic assays of the E142A mutant of Rv2002-M3 indicate that Glu-142 reverses the effect of Lys-157 in influencing the pKa of Tyr-153. This study suggests that the Rv2002 gene product is a unique member of the SDR family and is likely to be involved in steroid metabolism in M. tuberculosis. Our work demonstrates the power of the directed evolution technique as a general way of overcoming the difficulties in overexpressing the target proteins in soluble form. |
==About this Structure== | ==About this Structure== | ||
| - | 1NFF is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis Mycobacterium tuberculosis] with NAD as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/3-alpha-(or_20-beta)-hydroxysteroid_dehydrogenase 3-alpha-(or 20-beta)-hydroxysteroid dehydrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.53 1.1.1.53] Full crystallographic information is available from [http:// | + | 1NFF is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis Mycobacterium tuberculosis] with <scene name='pdbligand=NAD:'>NAD</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/3-alpha-(or_20-beta)-hydroxysteroid_dehydrogenase 3-alpha-(or 20-beta)-hydroxysteroid dehydrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.53 1.1.1.53] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1NFF OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Mycobacterium tuberculosis]] | [[Category: Mycobacterium tuberculosis]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Park, M | + | [[Category: Park, M S.]] |
| - | [[Category: Suh, S | + | [[Category: Suh, S W.]] |
| - | [[Category: TBSGC, TB | + | [[Category: TBSGC, TB Structural Genomics Consortium.]] |
| - | [[Category: Waldo, G | + | [[Category: Waldo, G S.]] |
| - | [[Category: Yang, J | + | [[Category: Yang, J K.]] |
[[Category: NAD]] | [[Category: NAD]] | ||
[[Category: directed evolution]] | [[Category: directed evolution]] | ||
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[[Category: tbsgc]] | [[Category: tbsgc]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:05:28 2008'' |
Revision as of 12:05, 21 February 2008
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Crystal structure of Rv2002 gene product from Mycobacterium tuberculosis
Overview
One of the serious bottlenecks in structural genomics projects is overexpression of the target proteins in soluble form. We have applied the directed evolution technique and prepared soluble mutants of the Mycobacterium tuberculosis Rv2002 gene product, the wild type of which had been expressed as inclusion bodies in Escherichia coli. A triple mutant I6TV47MT69K (Rv2002-M3) was chosen for structural and functional characterizations. Enzymatic assays indicate that the Rv2002-M3 protein has a high catalytic activity as a NADH-dependent 3alpha, 20beta-hydroxysteroid dehydrogenase. We have determined the crystal structures of a binary complex with NAD(+) and a ternary complex with androsterone and NADH. The structure reveals that Asp-38 determines the cofactor specificity. The catalytic site includes the triad Ser-140Tyr-153Lys-157. Additionally, it has an unusual feature, Glu-142. Enzymatic assays of the E142A mutant of Rv2002-M3 indicate that Glu-142 reverses the effect of Lys-157 in influencing the pKa of Tyr-153. This study suggests that the Rv2002 gene product is a unique member of the SDR family and is likely to be involved in steroid metabolism in M. tuberculosis. Our work demonstrates the power of the directed evolution technique as a general way of overcoming the difficulties in overexpressing the target proteins in soluble form.
About this Structure
1NFF is a Single protein structure of sequence from Mycobacterium tuberculosis with as ligand. Active as 3-alpha-(or 20-beta)-hydroxysteroid dehydrogenase, with EC number 1.1.1.53 Full crystallographic information is available from OCA.
Reference
Directed evolution approach to a structural genomics project: Rv2002 from Mycobacterium tuberculosis., Yang JK, Park MS, Waldo GS, Suh SW, Proc Natl Acad Sci U S A. 2003 Jan 21;100(2):455-60. Epub 2003 Jan 10. PMID:12524453
Page seeded by OCA on Thu Feb 21 14:05:28 2008
Categories: 3-alpha-(or 20-beta)-hydroxysteroid dehydrogenase | Mycobacterium tuberculosis | Single protein | Park, M S. | Suh, S W. | TBSGC, TB Structural Genomics Consortium. | Waldo, G S. | Yang, J K. | NAD | Directed evolution | Gfp | Hydroxysteroid dehydrogenase | Protein structure initiative | Psi | Sdr | Structural genomics | Tb structural genomics consortium | Tbsgc
