1nff

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spinqualitylabelsall models 1nff, resolution 1.80Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

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 NAD 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

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