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Human mevalonate diphosphate decarboxylase: Functional tests, structural progress, and assignments of active site residues
Author(s) -
Miziorko Henry M.,
Voynova Natalia E.,
Fu Zhuji,
Battaile Kevin,
Kim JungJa P.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.611.4
Subject(s) - mevalonate pathway , biochemistry , active site , enzyme , chemistry , binding site , transferase , stereochemistry , biology , reductase
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.