Identification and Functional Characterization of an Active-site Lysine in Mevalonate Kinase

Potter, David A.; Wojnar, Jean M.; Narasimhan, Chakravarthi; Miziorko, Henry M.

doi:10.1074/jbc.272.9.5741

Cited by 52 publications

(63 citation statements)

References 27 publications

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“…This shows that the HIDS mutations are evenly distributed along the coding region of MVK, which contrasts to that of mevalonic aciduria which essentially clusters between amino acids 243 and 334. Except for H20P 14,15 and some of the HIDS mutations are very close to these important amino acids.…”

Section: Position Of the Mutationsmentioning

confidence: 97%

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Molecular analysis of MVK mutations and enzymatic activity in hyper-IgD and periodic fever syndrome

et al. 2001

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Hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS) is an autosomal recessive inflammatory disorder characterised by recurrent episode of fever associated with lymphadenopathy, abdominal distress, joint involvement and skin lesions. We recently demonstrated that mutations in the mevalonate kinase gene (MVK) are associated with HIDS. Direct DNA sequencing was done to screen the entire coding region of MVK in 25 unrelated patients with HIDS. Mutations were detected in the coding region of the gene including 11 missense mutations, one deletion, the absence of expression of one allele, as well as three novel polymorphisms. Seven of these mutations are novel. The large majority of the patients were compound heterozygotes for two mutations. Of these, V377I (G?A) is the most common mutation occurring in 20 unrelated patients and was found to be associated with I268T in six patients. Mutations were associated with a decrease of mevalonate kinase (MK) (ATP:mevalonate 5-phosphotransferase, EC 2.7.I.36) enzymatic activity but not as profound as in mevalonic aciduria, a syndrome also caused by a deficient activity of MK. In HIDS the mutations are located all along the protein which is different from mevalonic aciduria where MK mutations are mainly clustered to a same region of the protein. On the basis of this study, we propose that the diagnostic screen of MVK in HIDS should be first directed on V377I and I268T mutations. Three patients are also described to illustrate the genotypic and phenotypic overlap with mevalonic aciduria. European Journal of Human Genetics (2001) 9, 260 ± 266.

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Section: Position Of the Mutationsmentioning

confidence: 97%

“…K13, E19, E193 and D204 are known important functional amino. 14,15 Deletion 1 refers to our previous report 4 and deletion 2 refers to this study. HIDS mutations are indicated in bold characters and mevalonic aciduria mutations (#) are summarised in the large square.…”

Section: Genotype-phenotype Correlationmentioning

confidence: 98%

Molecular analysis of MVK mutations and enzymatic activity in hyper-IgD and periodic fever syndrome

et al. 2001

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“…Mevalonate kinase is a member of GHMP kinase superfamily, a family of metabolic enzymes with a highly conserved glycine-rich motif (PXGXGLGSSAA) that is implicated in the binding of ATP (41)(42)(43). It might first appear that the presence of an ATP binding site in MVK might make it vulnerable for nonspecific binding to any RNA molecule due to the presence of adenine residues.…”

Section: Fig 6 Mevalonate Kinase-depleted Lrbp Preparation Shows Dementioning

confidence: 99%

Isolation and Characterization of a Novel trans-Factor for Luteinizing Hormone Receptor mRNA from Ovary

Nair

Menon

2004

Journal of Biological Chemistry

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Post-transcriptional mechanisms play a major role in regulating luteinizing hormone (LH) receptor mRNA expression in the ovary. An ovarian cytosolic protein that we have identified in rats and humans, which binds to a polypyrimidine-rich bipartitate sequence in the coding region of LHR mRNA, acts as a trans-acting factor in this process. In the present study, we isolated and characterized this LH receptor mRNA-binding protein (LRBP) from rat ovary. LRBP was purified to homogeneity by cation exchange chromatography followed by Northwestern analysis and subsequent elution of the single protein band from SDS-polyacrylamide gel. Purified LRBP was subjected to N-terminal microsequencing followed by homology search, which revealed its identity as mevalonate kinase. Purified rat mevalonate kinase antibody recognized the gel-purified LRBP on Western blots performed with one-and two-dimensional SDSpolyacrylamide gels. When recombinant mevalonate kinase produced in human embryonic kidney cells ( The interaction of luteinizing hormone (LH) 1 with its cell surface receptors controls reproductive functions including steroidogenesis in the gonad (1). In the ovary, luteinizing hormone receptor (LHR), a member of G s -protein coupled receptors, regulates ovarian function mainly through increased production of cAMP (2-4). The cell surface expression of LHR varies during different stages of the ovarian cycle. Its expression in granulosa cells and luteal cells is greatly decreased by an endogenous preovulatory LH surge or by the administration of a pharmacological dose of human chorionic gonadotropin (hCG), a placental counterpart of LH (5-8). We have shown that the decline in cell surface LHR expression seen after hCG administration is paralleled by a specific loss of all four LHR mRNA transcripts (6.7, 4.4, 2.6, and 1.8 kb) in the ovary (9). Furthermore, the loss of LHR mRNA does not result from decreased transcription but occurs post-transcriptionally with a 3-fold decrease in half-life (8).Regulation of mRNA turnover is one of the major control mechanisms of gene expression in all organisms. mRNA halflives are influenced by the interaction of various cytoplasmic proteins (trans-acting factors) with regulatory regions (cis-acting elements) in the mRNA, forming ribonucleoprotein (RNP) complexes (10). The formation and disruption of RNP complexes in response to various cellular stimuli mainly controls the turnover of cytoplasmic mRNA. Studies have indicated the presence of cis-acting regulatory elements in the 5Ј-untranslated region, coding region, and 3Ј-untranslated region of mRNA (10). A number of cytoplasmic trans-acting factors, some of which shuttle between the nucleus and cytoplasm, have been identified as mRNA-stabilizing, destabilizing, or translational repressor proteins (11-16). c-Fos, c-Myc, tropoelastin, thymidylate synthase, and dihydrofolate reductase are some of the mRNAs containing regulatory elements in the coding region for trans-acting factors (17-24).We have identified a LHR mRNA-binding protein in rat and hu...

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“…Experiments were performed analogously to earlier work, which demonstrated equilibrium binding of the spin-labeled probe to wild-type enzyme (6,7), except that 100 mM KCl was included in the sample buffer to minimize nonspecific binding. Using this probe, which contains a reporter group attached to the nucleotide's ␥-phosphoryl, binding is determined by measuring diminution of the amplitude of the signal attributable to unbound spin label.…”

Section: Mutagenic Substitution Of Alcohol Sidementioning

confidence: 99%

“…Only 5% of the amino acids that encode the human enzyme are invariant. Included in this select group are the residues (Lys-13, 2 Glu-193, Asp-204) that our laboratory has implicated previously (6,7) in various active site functions. Inspection of mevalonate kinase's amino acid sequence does not suggest that substrate ATP binding relies on well established consensus sequences such as Walker A or Walker B motifs.…”

mentioning

confidence: 99%

Investigation of Invariant Serine/Threonine Residues in Mevalonate Kinase

Cho

Rios

Kim

et al. 2001

Journal of Biological Chemistry

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Mevalonate kinase serine/threonine residues have been implicated in substrate binding and inherited metabolic disease. Alignment of >20 mevalonate kinase sequences indicates that Ser-145, Ser-146, Ser-201, and Thr-243 are the only invariant residues with alcohol side chains. These residues have been individually mutated to alanine. Structural integrity of the mutants has been demonstrated by binding studies using fluorescent and spin-labeled ATP analogs. Kinetic characterization of the mutants indicates only modest changes in K m(ATP) . K m for mevalonate increases by Ϸ20-fold for S146A, Ϸ40-fold for T243A, and 100-fold for S201A. V max changes for S145A, S201A, and T243A are <3-fold. Thus, the 65-fold activity decrease associated with the inherited human T243I mutation seems attributable to the nonconservative substitution rather than any critical catalytic function. V max for S146A is diminished by 4000-fold. In terms of V/K MVA , this substitution produces a 10 5 -fold effect, suggesting an active site location and catalytic role for Ser-146. The large k cat effect suggests that Ser-146 productively orients ATP during catalysis. K D(Mg-ATP) increases by almost 40-fold for S146A, indicating a specific role for Ser-146 in liganding Mg 2؉ -ATP. Instead of mapping within a proposed C-terminal ATP binding motif, Ser-146 is situated in a centrally located motif, which characterizes the galactokinase/homoserine kinase/ mevalonate kinase/phosphomevalonate kinase protein family. These observations represent the first functional demonstration that this region is part of the active site in these related phosphotransferases.Isoprenoid biosynthesis is accomplished by diverse pathways that use either acetyl-CoA (1) or glyceraldehyde 3-phosphate and pyruvate (2) as starting materials. Mevalonate kinase (E.C. 2.7.1.36) represents a distinctive component of the former pathway, which functions in eukaryotes, archaebacteria, and some eubacteria. In the mevalonate-mediated pathway for isoprenoid production, attention has long been focused on HMGCoA 1 reductase, which catalyzes a highly regulated step. The diversity of species in which mevalonate kinase functions is reflected in the high degree of heterology that is apparent upon comparison of deduced sequences for this enzyme. Only 5% of the amino acids that encode the human enzyme are invariant. Included in this select group are the residues (Lys-13, 2 Glu-193, Asp-204) that our laboratory has implicated previously (6, 7) in various active site functions. Inspection of mevalonate kinase's amino acid sequence does not suggest that substrate ATP binding relies on well established consensus sequences such as Walker A or Walker B motifs. However, two distinct glycine-rich stretches of amino acid sequence have been proposed as potential ATP-binding regions (8, 9), although no direct evidence that tests these hypotheses or discriminates between the two candidate sequences has appeared.For phosphotransferase enzymes, there are numerous examples of serines or threonines that map to the...

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Identification and Functional Characterization of an Active-site Lysine in Mevalonate Kinase

Cited by 52 publications

References 27 publications

Molecular analysis of MVK mutations and enzymatic activity in hyper-IgD and periodic fever syndrome

Molecular analysis of MVK mutations and enzymatic activity in hyper-IgD and periodic fever syndrome

Isolation and Characterization of a Novel trans-Factor for Luteinizing Hormone Receptor mRNA from Ovary

Investigation of Invariant Serine/Threonine Residues in Mevalonate Kinase

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