Staphylococcus aureus mevalonate kinase: isolation and characterization of an enzyme of the isoprenoid biosynthetic pathway

It has been proposed that isoprenoid biosynthesis in several gram-positive cocci depends on the mevalonate pathway for conversion of acetyl coenzyme A to isopentenyl diphosphate. Mevalonate kinase catalyzes a key reaction in this pathway. In this study the enzyme from Staphylococcus aureus was expressed in Escherichia coli, isolated in a highly purified form, and characterized. The overall amino acid sequence of this enzyme was very heterologous compared with the sequences of eukaryotic mevalonate kinases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration chromatography suggested that the native enzyme is a monomer with a molecular mass of approximately 33 kDa. The specific activity was 12 U/mg, and the pH optimum was 7.0 to 8.5. The apparent K(m) values for R,S-mevalonate and ATP were 41 and 339 micro M, respectively. There was substantial substrate inhibition at millimolar levels of mevalonate. The sensitivity to feedback inhibition by farnesyl diphosphate and its sulfur-containing analog, farnesyl thiodiphosphate, was characterized. These compounds were competitive inhibitors with respect to ATP; the K(i) values were 46 and 45 micro M for farnesyl diphosphate and its thio analog, respectively. Parallel measurements with heterologous eukaryotic mevalonate kinases indicated that S. aureus mevalonate kinase is much less sensitive to feedback inhibition (K(i) difference, 3 orders of magnitude) than the human enzyme. In contrast, both enzymes tightly bound trinitrophenyl-ATP, a fluorescent substrate analog, suggesting that there are similarities in structural features that are important for catalytic function.     

The activity and kinetic properties of mevalonate kinase in superovulated rat ovary

Methods are described for the assay and partial purification of mevalonate kinase from superovulated rat ovary. The total activity of mevalonate kinase in superovulated rat ovary was 1.6+/-0.14units/g wet wt.; it was unchanged by the administration of luteinizing hormone in vivo. The K(m) of a partially purified preparation of mevalonate kinase for dl-Mevalonate was 3.6+/-0.5mum; its K(m) for MgATP(2-) was 120+/-7.7mum. The enzyme was inhibited by geranyl pyrophosphate and farnesyl pyrophosphate, but not by isopentenyl pyrophosphate or 3,3'-dimethylallyl pyrophosphate. dl-mevalonate 5-phosphate inhibited at high concentrations. With both geranyl pyrophosphate and farnesyl pyrophosphate the inhibition was competitive with respect to MgATP(2-). The K(i) for inhibition by geranyl pyrophosphate was 1.3+/-0.2mum; the K(i) for inhibition by farnesyl pyrophosphate was 1.0+/-0.3mum. These findings are discussed with reference to the control by luteinizing hormone of steroidogenesis from acetate.     

3-Hydroxy-3-methylglutaryl CoA reductase and mevalonate kinase of Neurospora crassa

Two enzymes of polyisoprenoid synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (mevalonate:NADP oxidoreductase [acylating CoA], EC 1.1.1.34) and mevalonate kinase (ATP:mevalonate 5-phosphotransferase, EC 2.7.1.36), are present in the microsomal and soluble fractions of Neurospora crassa, respectively. HMG CoA reductase specifically uses NADPH as reductant and has a K(m) for dl-HMG CoA of 30 micro M. The activities of HMG CoA reductase and mevalonate kinase are low in conidia and increase threefold during the first 12 hr of stationary growth. Maximum specific activities of both enzymes occur when aerial hyphae and conidia first appear (2 days), but total activities peak later (3-4 days). Addition to the growth media of ergosterol or beta-carotene, alone or in combination, does not affect the specific or total activity of either enzyme. The mevalonate kinase of N. crassa, purified 200-fold to a specific activity of 5 micro moles/min/mg, is free from HMG CoA reductase, phosphomevalonate kinase, ATPase, adenylate kinase, and NADH oxidase activities. Mevalonate kinase specifically requires ATP as cosubstrate and exhibits a marked preference for Mg(2+) over Mn(2+), especially at high ratios of divalent metal ion to ATP. Kinase activity is inhibited by p-hydroxymercuribenzoate, and this inhibition is partially prevented by mevalonate or MgATP. Optimum activity occurs at pH 8.0-8.5 and at about 55 degrees C. The Neurospora kinase, like that of hog liver, has a sequential mechanism for substrate addition. The Michaelis constants obtained were 2.8 mM for dl-mevalonate and 1.8 mM for MgATP(-2). Geranyl pyrophosphate is an inhibitor competitive with MgATP (K(i) = 0.11 mM).     

Purification and characterization of mevalonate kinase from suspension-cultured cells of Catharanthus roseus (L.) G. Don

Mevalonate kinase was purified to homogeneity from Catharanthus roseus (L.) G. Don suspension-cultured cells. The purified enzyme had an M(r) of 104,600 and a subunit size of about 41,500. Kinetic studies indicated an ordered sequential mechanism of action, in which mevalonate was the first substrate to bind and ADP was the last product to leave the enzyme. True values for the kinetic constants were determined for mevalonate, with K(ma) = 76 microM and K(ia) = 74 microM, and for ATP, with K(mb) = 0.13 mM and K(ib) = 0. 13 mM; the true V(max) was calculated to be 138.7 nkat/mg of protein. Product inhibition was only detectable at rather high concentrations: above 0.7 mM for 5-phosphomevalonate and above 2 mM for ADP, with an ADP/ATP ratio of at least 1. Mevalonate kinase activity was shown to be strongly inhibited by farnesyl diphosphate. Farnesyl diphosphate acted as a competitive inhibitor toward ATP, with a K(i) value of 0.1 microM. Mevalonate kinase activity was dependent on the presence of divalent ions. At a concentration of 2 mM, Mg(2+) and Mn(2+) were best and equally effective in sustaining activity; compared to Mg(2+) and Mn(2+), relative activities of 35, 30, 16, 4.8, and 3.4% were detected at equimolar concentrations of Zn(2+), Fe(2+), Co(2+), Ca(2+), and Ni(2+), respectively. The pH-dependent activity profile of mevalonate kinase showed a broad pH optimum between pH 7 and 10, with a maximum at about pH 8.9.     

An improved purification procedure, an alternative assay and activation of mevalonate kinase by ATP

An improved procedure for the purification of pig liver mevalonate kinase (ATP:mevalonate 5-phosphotransferase, EC 2.7.1.36) is described. A high-voltage electrophoresis assay was developed for mevalonate kinase. The procedure separates mevalonate from phosphomevalonate and also from diphosphomevalonate so that it can be used to measure the subsequent enzyme, phosphomevalonate kinase (EC 2.7.4.2). The assay has allowed the reassessment of the metal ion and nucleotide specificity of the pig liver enzyme. Some of the previously reported properties reflected those of the enzymes in the coupling assay rather than mevalonate kinase itself. A series of compounds were tested as activators or inhibitors of mevalonate kinase. It was found that ATP4-, arsenate and, to a smaller extent, inorganic phosphate activated the enzyme. At fixed MgATP2- (1 mM) concentrations the activation of mevalonate kinase by free ATP4- at pH 8.0 was observed at concentrations at up to 10-fold that of MgATP2- before causing any inhibition. The presence of free ATP4- resulted in a biphasic Lineweaver-Burke plot with apparent Km values for MgATP2- being 0.14 mM and 60 microM, respectively. Fluorescence measurements were consistent with the notion that the binding of excess ATP4- to the enzyme caused a conformational change.     

Characterization of a feedback-resistant mevalonate kinase from the archaeon Methanosarcina mazei

The mevalonate pathway is utilized for the biosynthesis of isoprenoids in many bacterial, eukaryotic, and archaeal organisms. Based on previous reports of its feedback inhibition, mevalonate kinase (MVK) may play an important regulatory role in the biosynthesis of mevalonate pathway-derived compounds. Here we report the purification, kinetic characterization, and inhibition analysis of the MVK from the archaeon Methanosarcina mazei. The inhibition of the M. mazei MVK by the following metabolites derived from the mevalonate pathway was explored: dimethylallyl diphosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), isopentenyl monophosphate (IP), and diphosphomevalonate. M. mazei MVK was not inhibited by DMAPP, GPP, FPP, diphosphomevalonate, or IP, a proposed intermediate in an alternative isoprenoid pathway present in archaea. Our findings suggest that the M. mazei MVK represents a distinct class of mevalonate kinases that can be differentiated from previously characterized MVKs based on its inhibition profile.     

Expression of the yeast mevalonate kinase gene in trangenic tobacco

The coding region of the yeast mevalonate kinase gene (ERG12), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, has been inserted in tobacco (Nicotiana tabacum cv. Paraguay Bell) using an Agrobacterium tumefaciens binary vector system. Integration and expression of the ERG12 chimaeric gene was demonstrated in several independent transformants in which specific mevalonate kinase (MK) activity in young plantlets was increased by about 60% on average. The expression of this MK gene was accompanied by phenotypical modifications, such as acceleration of regenerating processes, lateral bud growth, and peculiar flowering behaviour. A higher chlorophyll content all along the plant development, paralleled by an unusual starch accumulation in the leaves of young plantlets and, later, in roots of full-grown plants, was also detected. Overexpression of the MK gene led also to a stronger inhibition of cytokinin-induced plant growth by methyl jasmonate in transgenic plants. All these events may be interpreted as a possible modification of the hormonal balance in transgenic tobaccos.     

Functional evaluation of conserved basic residues in human phosphomevalonate kinase

Phosphomevalonate kinase (PMK) catalyzes the cation-dependent reaction of mevalonate 5-phosphate with ATP to form mevalonate 5-diphosphate and ADP, a key step in the mevalonate pathway for isoprenoid/sterol biosynthesis. Animal PMK proteins belong to the nucleoside monophosphate (NMP) kinase family. For many NMP kinases, multiple basic residues contribute to the neutralization of the negatively charged pentacoordinate phosphate reaction intermediate. Loss of basicity can result in catalytically impaired enzymes. On the basis of this precedent, conserved basic residues of human PMK have been mutated, and purified forms of the mutated proteins have been kinetically and biophysically characterized. K48M and R73M mutants exhibit diminished Vmax values in both reaction directions (>1000-fold) with only slight Km perturbations (<10-fold). In both forward and reverse reactions, R110M exhibits a large (>10,000-fold) specific activity diminution. R111M exhibits substantially inflated Km values for mevalonate 5-phosphate and mevalonate 5-diphosphate (60- and 30-fold, respectively) as well as decreases [50-fold (forward) and 85-fold (reverse)] in Vmax. R84M also exhibits inflated Km values (50- and 33-fold for mevalonate 5-phosphate and mevalonate 5-diphosphate, respectively). The Ki values for R111M and R84M product inhibition by mevalonate 5-diphosphate are inflated by 45- and 63-fold; effects are comparable to the 30- and 38-fold inflations in Km for mevalonate 5-diphosphate. R141M exhibits little perturbation in Vmax [14-fold (forward) and 10-fold (reverse)] but has inflated Km values for ATP and ADP (48- and 136-fold, respectively). The Kd of ATP for R141M, determined by changes in tryptophan fluorescence, is inflated 27-fold compared to wt PMK. These data suggest that R110 is important to PMK catalysis, which is also influenced by K48 and R73. R111 and R84 contribute to binding of mevalonate 5-phosphate and R141 to binding of ATP.     

Investigation of invariant serine/threonine residues in mevalonate kinase. Tests of the functional significance of a proposed substrate binding motif and a site implicated in human inherited disease

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 approximately 20-fold for S146A, approximately 40-fold for T243A, and 100-fold for S201A. V(max) changes for S145A, S201A, and T243A are < or =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.     

Influence of mevalonate kinase on studies of the MgATP-dependent inactivator of 3-hydroxy-3-methylglutaryl coenzyme A reductase

The nature of the MgATP-dependent inactivator of 3-hydroxy-3-methylglutaryl coenzyme A reductase has been studied. Several observations suggest that reductase inactivator preparations from both microsomes and cytosol possess mevalonate kinase activity. (1) Reductase inactivator (reductase kinase) activity copurified with mevalonate kinase activity. (2) Inactivator activity was inhibited by geranyl pyrophosphate and farnesyl pyrophosphate, known to be potent inhibitors of mevalonate kinase. (3) Addition of an excess of mevalonate completely prevented inhibition of reductase activity. (4) Formation of phosphomevalonate fully accounted for the decreased amount of mevalonate formed in the presence of inactivator and MgATP. (5) When reductase activity was measured by NADPH oxidation, no inhibition was observed. Clearly, the presence of mevalonate kinase in reductase inactivator preparations can lead to misinterpretations concerning whether reductase activity is regulated by phosphorylation-dephosphorylation. In this paper, we present several methods and approaches which can be used to critically evaluate this possibility.     

Biochemical and genetic aspects of mevalonate kinase and its deficiency

Mevalonate kinase (MK) is an essential enzyme in the mevalonate pathway which produces numerous cellular isoprenoids. The enzyme has been characterized both at the biochemical and the molecular level in a variety of organisms. Despite the fact that mevalonate kinase is not the rate-limiting enzyme in isoprenoid biosynthesis, its activity is subject to feedback regulation by the branch-point intermediates geranyldiphosphate, farnesyldiphosphate and geranylgeranyldiphosphate. Recently, the importance of mevalonate kinase was demonstrated by the identification of its deficiency as the biochemical and molecular cause of the inherited human disorders mevalonic aciduria and hyperimmunoglobulinemia D and periodic fever syndrome. The pathophysiology of these disorders is not yet understood, but eventually will give insight into the in vivo role of mevalonate kinase and isoprenoid biosynthesis with respect to the acute phase response and fever. The subcellular localization of mevalonate kinase is still a matter of debate. The enzyme could be localized predominantly in the cytosol, or in peroxisomes, or it is associated differentially with peroxisomes. Here we review the biochemical and molecular properties of MK, and discuss its biological significance, the regulation of its enzyme activity and finally its subcellular localization.     

Discovery and characterization of a substrate selective p38alpha inhibitor

A novel inhibitor of p38 mitogen-activated protein kinase (p38), CMPD1, identified by high-throughput screening, is characterized herein. Unlike the p38 inhibitors described previously, this inhibitor is substrate selective and noncompetitive with ATP. In steady-state kinetics experiments, CMPD1 was observed to prevent the p38alpha-dependent phosphorylation (K(i)(app) = 330 nM) of the splice variant of mitogen-activated protein kinase-activated protein kinase 2 (MK2a) that contains a docking domain for p38alpha and p38beta, but it did not prevent the phosphorylation of ATF-2 (K(i)(app) > 20 microM). In addition to kinetic studies, isothermal titration calorimetry and surface plasmon resonance experiments were performed to elucidate the mechanism of inhibition. While isothermal titration calorimetry analysis indicated that CMPD1 binds to p38alpha, CMPD1 was not observed to compete with ATP for p38alpha, nor was it able to interrupt the binding of p38alpha to MK2a observed by surface plasmon resonance. Therefore, deuterium exchange mass spectrometry (DXMS) was employed to study the p38alpha.CMPD1 inhibitory complex, to provide new insight into the mechanism of substrate selective inhibition. The DXMS data obtained for the p38alpha.CMPD1 complex were compared to the data obtained for the p38alpha.MK2a complex and a p38alpha.active site binding inhibitor complex. Alterations in the DXMS behavior of both p38alpha and MK2a were observed upon complex formation, including but not limited to the interaction between the carboxy-terminal docking domain of MK2a and its binding groove on p38alpha. Alterations in the D(2)O exchange of p38alpha produced by CMPD1 suggest that the substrate selective inhibitor binds in the vicinity of the active site of p38alpha, resulting in perturbations to regions containing nucleotide binding pocket residues, docking groove residues (E160 and D161), and a Mg(2+) ion cofactor binding residue (D168). Although the exact mechanism of substrate selective inhibition by this novel inhibitor has not yet been disclosed, the results suggest that CMPD1 binding in the active site region of p38alpha induces perturbations that may result in the suboptimal positioning of substrates and cofactors in the transition state, resulting in selective inhibition of p38alpha activity.     

Catalytically active MAP KAP kinase 2 structures in complex with staurosporine and ADP reveal differences with the autoinhibited enzyme

MAP KAP kinase 2 (MK2), a Ser/Thr kinase, plays a crucial role in the inflammatory process. We have determined the crystal structures of a catalytically active C-terminal deletion form of human MK2, residues 41-364, in complex with staurosporine at 2.7 A and with ADP at 3.2 A, revealing overall structural similarity with other Ser/Thr kinases. Kinetic analysis reveals that the K(m) for ATP is very similar for MK2 41-364 and p38-activated MK2 41-400. Conversely, the catalytic rate and binding for peptide substrate are dramatically reduced in MK2 41-364. However, phosphorylation of MK2 41-364 by p38 restores the V(max) and K(m) for peptide substrate to values comparable to those seen in p38-activated MK2 41-400, suggesting a mechanism for regulation of enzyme activity.     

Expression and purification of Arg196 and Lys272 mutants of mevalonate kinase from Methanococcus jannaschii

In microorganisms and plants, mevalonate kinase is involved in the biosynthesis of isoprenoid derivatives, one of the largest groups of natural products. We subcloned the gene of mevalonate kinase from Methanococcus jannaschii into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5' end of the gene. A variety of mutant expression plasmids including pMMK(R196K), pMMK(R196Q), pMMK(R196V), pMMK(K272R), and pMMK(K272A) have been constructed using site-directed mutagenesis. The wild-type protein and mutants were overexpressed and purified with a nickel HiTrap chelating metal affinity column to homogeneity. CD spectroscopy of wild-type protein and mutants indicates that none of the above mutations induces significant secondary structural changes. The results from kinetic studies showed that Arg196 is an essential residue for the function of the enzyme. Kinetic studies of Lys272 mutants indicate that salt bridge Lys272-Glu14 plays an important role in maintaining the active site microenvironment that is essential for catalytic activity of the enzyme.     

Sites of control of hepatic cholesterol biosynthesis

An inhibition in the conversion of mevalonate to cholesterol has been demonstrated in liver of cholesterol-fed rats by both in vitro and in vivo methods. Synthesis decreased to 30% of the control value after 1 week and 20% after 1 month on a 1% cholesterol diet. After a year, synthesis from mevalonate was almost completely inhibited. The rate of conversion of squalene to cholesterol was not consistently decreased but that of farnesyl pyrophosphate to cholesterol was decreased considerably. The rate of conversion of mevalonate to farnesyl pyrophosphate by a soluble liver enzyme preparation was also decreased in cholesterol-fed animals. Sites of inhibition of cholesterol synthesis were detected before mevalonate, between mevalonate and farnesyl pyrophosphate, and after farnesyl pyrophosphate, probably at the conversion of farnesyl pyrophosphate to squalene. The inhibition of mevalonate conversion to cholesterol developed more slowly than that of acetate and appeared to be secondary to it. The maximum capacities of normal liver homogenates and slices to synthesize cholesterol from mevalonate were shown to be far greater than from acetate. Consequently, sites of inhibition after mevalonate probably do not have a significant effect on the over-all rate of cholesterol synthesis in the intact cholesterol-fed animal.     

Substrate binding order in mevalonate 5-diphosphate decarboxylase from chicken liver

The substrate binding order of chicken liver mevalonate 5-diphosphate decarboxylase was investigated by using competitive inhibitors of the substrates. Mevalonate and mevalonate 5-phosphate showed mixed inhibition when ATP was the varied substrate. Both analogues of ATP, adenosine 5'-O-(3-thiotriphosphate) and beta-tau methylene adenosine 5'-triphosphate showed uncompetitive inhibition against mevalonate 5-diphosphate. These results are in accordance with an ordered sequential mechanism with mevalonate 5-diphosphate as the first substrate to bind to the enzyme.     

Identification of a bisphosphonate that inhibits isopentenyl diphosphate isomerase and farnesyl diphosphate synthase.

We and others have recently shown that the major molecular target of nitrogen-containing bisphosphonate drugs is farnesyl diphosphate synthase, an enzyme in the mevalonate pathway. In an in vitro screen, we discovered a bisphosphonate, NE21650, that potently inhibited farnesyl diphosphate synthase but, unlike other N-BPs investigated, was also a weak inhibitor of isopentenyl diphosphate isomerase. NE21650 was a more potent inhibitor of protein prenylation in osteoclasts and macrophages, and a more potent inhibitor of bone resorption in vitro, than alendronate, despite very similar IC(50) values for inhibition of farnesyl diphosphate synthase. Our observations show that minor changes to the structure of bisphosphonates allow inhibition of more than one enzyme in the mevalonate pathway and suggest that loss of protein prenylation due to inhibition of more than one enzyme in the mevalonate pathway may lead to an increase in antiresorptive potency compared to bisphosphonates that only inhibit farnesyl diphosphate synthase.     

Modulation of collagen gel compaction by extracellular ATP is MAPK and NF-κB pathways dependent

Understanding the mechanisms that regulate mechanosensitivity in osteoblasts is important for controlling bone homeostasis and the development of new drugs to combat bone loss. It is believed that prestress or force generation (the tensile stress within the cell body) plays an important role in regulating cellular mechanosensitivity. In the present study, a three-dimensional (3D) collagen culture was used to monitor the change in prestress of the osteoblast-like cells. Collagen hydrogel compaction has been used as an indicator of the change in the degree of cell prestress. Previous results in this model demonstrated that extracellular ATP reduced the mechanosensitivity of osteoblasts by reducing cellular prestress. To elucidate the potential mechanisms involved in this process, the signaling pathways downstream of P2 purinoceptors involved in regulating the compaction of type I collagen gels were investigated. By using specific inhibitors to these signaling pathways, we found that ATP-induced reduction in collagen gel compaction rate is dependent on mitogen-activated protein kinase (MAKP) and NF-κB pathways. However, blocking protein kinase C with GF109203X did not change the compaction kinetics in the presence of ATPγS. Moreover, blocking cyclic AMP (cAMP), phosphatidylinositol-3 kinase (PI3K), calmodulin (CaM) or L-type voltage sensitive calcium channels did not affect ATP's ability to reduce collagen gel compaction. The results from the present and previous studies indicate that extracellular ATP may act as a negative feedback modulator in the mechanotransduction system since mechanical stimuli increase ATP release from stimulated cells.     

Crystal structure of the Streptococcus pneumoniae mevalonate kinase in complex with diphosphomevalonate

Streptococcus pneumoniae, a ubiquitous gram-positive pathogen with an alarming, steadily evolving resistance to frontline antimicrobials, poses a severe global health threat both in the community and in the clinic. The recent discovery that diphosphomevalonate (DPM), an essential intermediate in the isoprenoid biosynthetic pathway, potently and allosterically inhibits S. pneumoniae mevalonate kinase (SpMK) without affecting the human isozyme established a new target and lead compound for antimicrobial design. Here we present the crystal structure of the first S. pneumoniae mevalonate kinase, at a resolution of 2.5 A and in complex with DPM.Mg(2+) in the active-site cleft. Structural comparison of SpMK with other members of the GHMP kinase family reveals that DPM functions as a partial bisubstrate analog (mevalonate linked to the pyrophosphoryl moiety of ATP) in that it elicits a ternary-complexlike form of the enzyme, except for localized disordering in a region that would otherwise interact with the missing portion of the nucleotide. Features of the SpMK-binding pockets are discussed in the context of established mechanistic findings and inherited human diseases linked to MK deficiency.