Human mevalonate diphosphate decarboxylase: Functional tests, structural progress, and assignments of active site residues

Expression in E. coli of his‐tagged human mevalonate diphosphate decarboxylase (hMDD) has expedited enzyme isolation, characterization, functional investigation, and crystallization. hMDD exhibits Vm = 6 U/mg; Km for ATP is 0.7 mM; Km for (R,S) mevalonate diphosphate is 29 uM. Diphosphoglycolyl proline is a competitive inhibitor (Ki = 2 uM) against MPP. Several conserved polar residues that, based on sequence alignment and structural overlay of yeast MDD and a mevalonate kinase‐ATP complex, are near the active site were mutated to test functional importance. R161Q exhibits ∼1000‐fold diminution in specific activity, although it binds fluorescent substrate analog, TNP‐ATP, like wild‐type enzyme. N17A exhibits a 50‐fold inflation in Km for mevalonate diphosphate. Crystallized hMDD (P2(1); a=87.0, b=53.2, c=97.8 Å, and beta=107.3°) diffracts to 2.4Å. Molecular replacement methods produced an initial structural model; refinement is in progress. Based on structural information, R161 and N17 map in the interior of the active site cleft. Predicted proximity (∼3 Å) of R161 to N17 and functional characterization of R161Q and N17A are compatible with their roles in binding/orientation of mevalonate diphosphate.