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Directed evolution approach to a structural genomics project: Rv2002 from Mycobacterium tuberculosis.
Jin Kuk Yang1, Min S Park, Geoffrey S Waldo
1Structural Proteomics Laboratory, School of Chemistry and Molecular Engineering, Seoul National University, Seoul 151-742, South Korea.
Summary
Directed evolution enabled soluble expression of Mycobacterium tuberculosis Rv2002 protein. The resulting mutant exhibits high 3alpha, 20beta-hydroxysteroid dehydrogenase activity, revealing structural insights into steroid metabolism.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Biology
Background:
- Protein overexpression in soluble form is a challenge in structural genomics.
- The Mycobacterium tuberculosis Rv2002 gene product, wild type, expressed as inclusion bodies.
Purpose of the Study:
- To overcome overexpression difficulties using directed evolution.
- To characterize soluble mutants of Rv2002 for structural and functional insights.
- To investigate the role of Rv2002 in M. tuberculosis steroid metabolism.
Main Methods:
- Directed evolution to generate soluble protein mutants.
- Protein expression in Escherichia coli.
- Enzymatic assays for catalytic activity.
- X-ray crystallography for structural determination (binary and ternary complexes).
Main Results:
- A triple mutant (Rv2002-M3) showed high NADH-dependent 3alpha, 20beta-hydroxysteroid dehydrogenase activity.
- Crystal structures revealed Asp-38 for cofactor specificity and a catalytic triad (Ser-140, Tyr-153, Lys-157).
- An unusual Glu-142 residue was identified, influencing Tyr-153 pKa, suggesting a unique SDR family member.
Conclusions:
- Rv2002 is a unique SDR family member involved in M. tuberculosis steroid metabolism.
- Directed evolution is a powerful tool for obtaining soluble proteins for structural and functional studies.
- The study provides structural and functional characterization of a key enzyme in mycobacterial steroid metabolism.