Structural and kinetic studies to show the importance of residues involved in flap domain movement and catalysis in HMG-COA reductase from Pseudomonas mevalonii

Moumita Sen, Purdue University

Abstract

Many enzymes have been described that have an “active site lid” also known in some proteins as a “flap”. This flap undergoes a conformational change from an open to a closed state in which it closes over the active site and protects it from the solvent. The conformational change that occurs is a rigid-body hinge-type motion. HMG-CoA is an example of such enzymes, in which the flap domain consists of three helices. HMG-CoA reductase is a metabolic enzyme that catalyzes the reduction of HMG-CoA to mevalonate. The present structural and kinetic study was performed on HMG-CoA reductase from Pseudomonas mevalonii. HMG-CoA reductase from this bacterium is easy to handle crystallographically, as the crystals are robust and can be incubated with various combinations of substrates and co-factors, in some cases resulting in the closing of the flap domain (the apoenzyme has an open flap domain). In this study three residues thought to be important in the movement of the flap domain of HMG-CoA reductase from P. mevalonii were mutated, and the mutants were studied using kinetic analysis and X-ray crystallography. Asn 188, Ile 377 and Thr 374 were the residues studied. Asn 188 is a residue involved in a hydrogen-bonding network that also involves CoA, NAD+ and a key catalytic residue from the flap domain. Mutation of this residue to alanine resulted in severe reduction in kcat/ Km, but no change in the Km for any of the substrates or the co-factors. None of the crystal structures of this mutant showed a closed flap domain. These results indicate that flap domain closure and hence the catalytic activity is affected in this mutant. The side chain of Ile 377 from the flap domain fits in a hydrophobic pocket formed by hydrophobic groups of different parts from the rest of the protein. Mutation of this residue to aspartic acid and to asparagine severely affected the catalytic activity, mutation to alanine somewhat affected the catalytic activity, whereas mutation to leucine hardly affected the activity. None of the X-ray structures of the I377A showed a closed flap, whereas, one structure of I377L showed a closed flap. These outcomes imply that the hydrophobicity of the side-chain of this residue is very important for flap domain closure and consequently the catalysis. Thr 374 is a part of the hinge of the motion of the flap domain. Its backbone psi angle undergoes a huge change between the closed and the open states of the flap domain. Mutation of this residue to proline, whose backbone movement is restricted, hugely affected the kcat/ Km but not the Km values of the protein. Also, this mutant failed to crystallize. Mutation of Thr 374 to alanine, which changed the side-chain without restricting the backbone movement, changed neither the kinetic nor the crystallographic behavior of the protein. These results point to the importance of the freedom of the backbone movement of this residue.

Degree

Ph.D.

Advisors

Stauffacher, Purdue University.

Subject Area

Biochemistry|Biophysics

COinS