Mevalonate is a key intermediate, and mevalonate kinase a key early enzyme, in isoprenoid and sterol synthesis. Mevalonate kinase deficiency caused by mutation of this gene results in mevalonic aciduria, a disease characterized psychomotor retardation, failure to thrive, hepatosplenomegaly, anemia and recurrent febrile crises.
Defects in this gene also cause hyperimmunoglobulinaemia D and periodic fever syndrome, a disorder characterized by recurrent episodes of fever associated with lymphadenopathy, arthralgia, gastrointestinal dismay and skin rash. All of these conserved residues are located in a region that defines the active site cavity Fig.
For example, the residues corresponding to motif III form a Gly-rich loop very close to the phosphate-binding loop corresponding to motif II. The relative orientation of these two residues as well as a continuous electron density connecting their side chains suggest a disulfide bond, despite the existence of dithiothreitol in the crystallization condition.
Formation of the disulfide linkage may stabilize the conformation of the putative phosphate-binding loop. In addition, Lys in the Gly-rich loop is 3. A dioxane molecule is bound about 3. In various HSK-substrate complex structures, this region is occupied by phosphate groups and hydrogen bonds with the main chain amides on the phosphate-binding loop are present. Three representative growth patterns are shown in the figure.
The PduX variants displaying these patterns are described in the text. Solid diamond, wild type; open triangle, SA; solid square, SA. The first group uses an ordered mechanism in which ATP is the first substrate to bind and includes galactokinase from rat liver 44 , yeast 45 , and human 46 and homoserine kinases from E. The second group appears to have an ordered mechanism in which the non-ATP substrate binds first and includes broad bean galactokinase 49 and hog liver mevalonate kinase The last group was shown to proceed by a random ternary complex mechanism such as E.
It is interesting that GHMP family members use different kinetic mechanisms despite the existence of highly similar tertiary structures. This suggests that this superfamily interacts with their substrates using analogous chemical and structural features but that their dynamics are quite different. The studies of PduX l-Thr kinase presented here are consistent with an ordered ternary complex mechanism in which ATP binds first and l-Thr binds second.
The most likely explanation for an ordered mechanism is that the binding of the first substrate induces a conformational change in the enzyme resulting in the formation of a high affinity binding site for the second substrate.
Prior studies of GHMP family members indicate critical catalytic and substrate binding roles for carboxylate- and hydroxyl-containing side chains 37 , 53 — Here we changed 10 highly conserved amino acids of PduX that have such side chains and tested the effect of these mutations on structure and kinetic parameters. Description This entry represents a group of predicted phosphomevalonate kinases from bacteria.
Phosphomevalonate kinase EC This pathway starts with three molecules of acetyl-CoA, which, in a series of six different enzyme reactions, are converted to isopentanyl pyrophosphate IPP , the basic C5 isoprene unit and a common intermediate for a number of pathways including isoprenoid and cholesterol biosynthesis.
There are two unrelated types of PMK.Here we changed 10 highly conserved amino acids of PduX that have such side chains and tested the effect of these mutations on structure and kinetic parameters. For example, the residues corresponding to motif III form a Gly-rich loop very close to the phosphate-binding loop corresponding to motif II. Because alanine has a small side chain without any charge or polarity, we made the isosteric substitution of glutamine for Glu Although the exact amino acids are somewhat different, the similarity of the molecular surfaces and the constellation of protein atoms in this region suggest that it is likely that in MVK these corresponding residues may form similar interactions with the purine ring. The phosphate-binding loop might be the main force to bind and stabilize the phosphate tail of ATP.
There are three additional helices I, J, and K that are considered to be an insert between strand h and helix L 4. Hence we propose a critical role of the hydroxyl group of Ser in catalysis of the PduX reaction. Nonorthologous gene displacement of phosphomevalonate kinase. The studies of PduX l-Thr kinase presented here are consistent with an ordered ternary complex mechanism in which ATP binds first and l-Thr binds second. Replacement of Asp with alanine or asparagine did not cause a significant change in kinetic parameters.
Our alignment results showed that there are four highly conserved motifs in PduX homologues.
It forms a distinct ATP binding loop not found in other kinase families
All other hits possessed Z-scores less than 5. Based on the analysis of the active cavity, the best candidate for the catalytic residue is Asp located about 4. A similar pocket is apparent in MVK that likely serves the same function. Protein Sci.
A conserved lysine residue near the acceptor hydroxyl group is proposed to lower its pKa facilitating proton abstraction 15 , 17 , 18 , Hence the mechanism of PduX may be more like that of homoserine kinase that is proposed to increase catalysis primarily by transition state stabilization