Biochemical and Structural Basis for Feedback Inhibition of Mevalonate Kinase and Isoprenoid Metabolism^,

Abstract

Mevalonate kinase (MK), which catalyzes a key reaction in polyisoprenoid and sterol metabolism in many organisms, is subject to feedback regulation by farnesyl diphosphate and related compounds. The structures of human mevalonate kinase and a binary complex of the rat enzyme incubated with farnesyl thiodiphosphate (FSPP) are reported. Significant FSPP hydrolysis occurs under crystallization conditions; this results in detection of farnesyl thiophosphate (FSP) in the structure of the binary complex. Farnesyl thiodiphosphate competes with substrate ATP to produce feedback inhibition of mevalonate kinase. The binding sites for these metabolites overlap, with the phosphate of FSP nearly superimposed on ATP's beta-phosphate and FSP's polyisoprenoid chain overlapping ATP's adenosine moiety. Several hydrophobic amino acid side chains are positioned near the polyisoprenoid chain of FSP and their functional significance has been evaluated in mutagenesis experiments with human MK, which exhibits the highest reported sensitivity to feedback inhibition. Results suggest that single and double mutations at T104 and I196 produce a significant inflation of the K(i) for FSPP (approximately 40-fold for T104A/I196A). Such an effect persists when K(i) values are normalized for effects on the K(m) for ATP, suggesting that it may be possible to engineer MK proteins with altered sensitivity to feedback inhibition. Comparison of animal MK protein alignments and structures with those of a MK protein from Streptococcus pneumoniae indicates that sequence differences between N- and C-terminal domains correlate with differences in interdomain angles. Bacterial MK proteins exhibit more solvent exposure of feedback inhibitor binding sites and, consequently, weaker binding of these inhibitors.

Quantification of mevalonate-5-phosphate using UPLC-MS/MS for determination of mevalonate kinase activity

“…Above these levels, the concentrations of Mg 2+ and ATP had no influence on the velocity of the enzyme. The K m [MVA] of 0.027 mmol/L determined by the method as presented here is comparable to the previously reported values for human recombinant protein [19][20][21], as well as to the values detected in human fibroblast [12,22].…”

The Structure and Dynamics of BmR1 Protein from Brugia malayi: In Silico Approaches

“…In comparative modeling by MODELLER 9v9 [10], a total of 250 initial models were generated from multiple templates (PDB id: 2G3Y, 2IE8 [72], 3QOE [73], 1V32, 2E87, 2R3V [74] and 3I4Q), followed by secondary structure restraints and loop refinement using MODELLER 9v9 [10]. DOPE score and molpdf were used to evaluate the models.…”

Characterization of a Feedback-Resistant Mevalonate Kinase from the Archaeon Methanosarcina mazei

“…The catalytic activities of the MVKs were measured using a modified spectrophotometric assay that couples ADP formation to pyruvate synthesis and reduction to lactate (13). The initial rate of disappearance of NADH serves as a measure of the phosphorylation of mevalonate by MVK.…”

“…Organisms manage these tasks through the intricate regulation of isoprenoid-producing pathways (15). MVK was demonstrated to be an important regulatory point in the mevalonate pathway in both bacteria (1,2,31) and eukaryotes (4,9,13,16,18). Previously the small-molecule regulation of MVKs could be divided into two classes.…”