Vitamin K-dependent Carboxylase Research Articles

Pathway to malonic acid-containing metabolites

A crucial malonic acid moiety in the antibiotic malonomycin has been shown to be installed by a bacterial vitamin K-dependent (VKD) carboxylase orthologue.

A vitamin K-dependent carboxylase orthologue is involved in antibiotic biosynthesis

Vitamin K-dependent carboxylase (VKDC) enzymes modify glutamate residues in mammalian vitamin K-dependent proteins, generating γ-carboxyglutamic acids with malonate moieties that mediate important physiological responses such as blood coagulation. Proteins with sequence similarity to mammalian VKDC are also found in bacteria; however, their function remains unknown. The antibiotic malonomycin from Streptomyces rimosus contains an unusual malonate group, of unknown origin, that is essential for its biological activity. Here, we show that a bacterial VKDC orthologue (MloH) is responsible for the malonic acid moiety in malonomycin. Using CRISPR/Cas9 gene editing, complementation and mutagenesis experiments, this VKDC-like enzyme was shown to α-carboxylate an aspartyl residue within a hybrid polyketide–nonribosomal peptide intermediate during malonomycin biosynthesis. This study reveals a highly unusual biosynthetic pathway to malonic acid-containing metabolites, providing a functional role for VKDC-like proteins in prokaryotes and a vitamin K-dependent carboxylation reaction with a non-proteinogenic substrate. The function of putative bacterial vitamin K-dependent carboxylases (VKDCs) has so far been uncertain. Now, Micklefield and co-workers show that a bacterial VKDC orthologue is involved in the biosynthesis of the antibiotic malonomycin, generating an unusual malonic acid moiety that is essential for its biological activity.

Vitamin K oxygenation, glutamate carboxylation, and processivity: defining the three critical facets of catalysis by the vitamin K-dependent carboxylase.

The vitamin K-dependent carboxylase uses vitamin K oxygenation to drive carboxylation of multiple glutamates in vitamin K-dependent proteins, rendering them active in a variety of physiologies. Multiple carboxylations of proteins are required for their activity, and the carboxylase is processive, so that premature dissociation of proteins from the carboxylase does not occur. The carboxylase is unique, with no known homology to other enzyme families, and structural determinations have not been made, rendering an understanding of catalysis elusive. Although a model explaining the relationship of oxygenation to carboxylation had been developed, until recently almost nothing was known of the function of the carboxylase itself in catalysis. In the past decade, discovery and analysis of naturally occurring carboxylase mutants has led to identification of functionally relevant residues and domains. Further, identification of nonmammalian carboxylase orthologs has provided a basis for bioinformatic analysis to identify candidates for critical functional residues. Biochemical analysis of rationally chosen carboxylase mutants has led to breakthroughs in understanding vitamin K oxygenation, glutamate carboxylation, and maintenance of processivity by the carboxylase. Protein carboxylation has also been assessed in vivo, and the intracellular environment strongly affects carboxylase function. The carboxylase is an integral membrane protein, and topological analysis, coupled with biochemical determinations, suggests that interaction of the carboxylase with the membrane is an important facet of function. Carboxylase homologs, likely acquired by horizontal transfer, have been discovered in some bacteria, and functional analysis of these homologs has the potential to lead to the discovery of new roles of vitamin K in biology.

The Vitamin K-dependent Carboxylase Generates γ-Carboxylated Glutamates by Using CO2 to Facilitate Glutamate Deprotonation in a Concerted Mechanism That Drives Catalysis

The γ-glutamyl carboxylase converts Glu to carboxylated Glu (Gla) to activate a large number of vitamin K-dependent proteins with diverse functions, and this broad physiological impact makes it critical to understand the mechanism of carboxylation. Gla formation is thought to occur in two independent steps (i.e. Glu deprotonation to form a carbanion that then reacts with CO(2)), based on previous studies showing unresponsiveness of Glu deprotonation to CO(2). However, our recent studies on the kinetic properties of a variant enzyme (H160A) showing impaired Glu deprotonation prompted a reevaluation of this model. Glu deprotonation monitored by tritium release from the glutamyl γ-carbon was dependent upon CO(2), and a proportional increase in both tritium release and Gla formation occurred over a range of CO(2) concentrations. This discrepancy with the earlier studies using microsomes is probably due to the known accessibility of microsomal carboxylase to water, which reprotonates the carbanion. In contrast, tritium incorporation experiments with purified carboxylase showed very little carbanion reprotonation and consequently revealed the dependence of Glu deprotonation on CO(2). Cyanide stimulated Glu deprotonation and carbanion reprotonation to the same extent in wild type enzyme but not in the H160A variant. Glu deprotonation that depends upon CO(2) but that also occurs when water or cyanide are present strongly suggests a concerted mechanism facilitated by His-160 in which an electrophile accepts the negative charge on the developing carbanion. This revised mechanism provides important insight into how the carboxylase catalyzes the reaction by avoiding the formation of a high energy discrete carbanion.

The effects of salicylate on enzymes of vitamin K metabolism.

The mechanism of salicylate-induced hypoprothrombinaemia has been investigated in the rat. Salicylate administration produced an increase in the percentage of the total liver vitamin that was present as vitamin K 2,3-epoxide, but the addition of salicylate did not influence vitamin K epoxide reductase activity in-vitro. Neither did it influence vitamin K-dependent carboxylase or vitamin K epoxidase activity. Both cytosolic and microsomal DT-diaphorase activities were, however, inhibited about 50% by 75 microM sodium salicylate. Salicylate inhibition was also observed when vitamin K quinone and NADH or dithiothreitol were used to support carboxylation. To achieve 50% inhibition required 0.5 mM salicylate with NADH as a reductant and 4 mM salicylate when dithiothreitol was the reductant. These results suggest that the main effect of salicylate on vitamin K metabolism is to inhibit quinone reductases and may be useful in explaining the inhibition of the biosynthesis of vitamin K-dependent clotting factors that occurs in salicylate-induced hypothrombinaemia. These data also demonstrate that the percentage of total liver vitamin present as vitamin K epoxide can be increased by agents that do not have a direct effect on the vitamin K epoxide reductase in-vitro.

Bioreductive Activation‐independent Delivery of Menahydroquinone‐4 via Prodrug and Its Action in Warfarin‐poisoned Rat Liver

The ester prodrugs of menahydroquinone-4 (menahydroquinone), the active form of menaquinone-4 (menaquinone, vitamin K2(20)), were identified in previous reports as prodrugs that could achieve the systemic liver-specific delivery of menahydroquinone. The present study was undertaken to investigate the mechanism of the prodrugs for vitamin K-dependent carboxylation and their action in the warfarin-poisoned rat liver. A rat liver microsomal test system for assessing the carboxylation mechanism of the prodrugs was developed. In this system, the pathways for cleavage (hydrolysis) of the prodrug and for the reductive activation of menaquinone can be provided, and the vitamin K-dependent carboxylase can accept the synthetic tripeptide BOC-Glu-Glu-Leu-OMe as substrate. With this system, the carboxylation stimulated with the prodrugs and the enzymatic cleavage of the prodrugs were determined. The prodrug could stimulate the carboxylase activity in the absence of dithiothreitol, an artificial activator of the reductive activation pathway of menaquinone, and the activity order was as follows: 1-monoester > 1,4-bisester > 4-monoester. The carboxylation activities of the prodrugs were in the same order as the liver microsomal enzymatic reconversion characteristics of the prodrugs. The carboxylation activity of the selected prodrug (1-monoester) was strongly inhibited in the presence of eserine, a carboxylesterase inhibitor. The prodrug could also stimulate the carboxylase under warfarin-poisoning conditions, where the vitamin K cycle was strongly inhibited. The results confirmed that the prodrugs could generate menahydroquinone in the active site, and that the resultant menahydroquinone could act as cofactor for the carboxylase without reductive activation processes of menaquinone to menahydroquinone. Such bioreductive activation-independent vitamin K-dependent carboxylation characteristics of the prodrugs leads to enhanced pharmacological efficacy in the treatment of hypoprothrombinaemia induced in patients undergoing coumarin and cephalosporin therapies.

High-level expression of functional recombinant human coagulation factor VII in insect cells

Recombinant coagulation factor VII (FVII) is used as a potential therapeutic intervention in hemophilia patients who produce antibodies against the coagulation factors. Mammalian cell lines provide low levels of expression, however, the Spodoptera frugiperda Sf9 cell line and baculovirus expression system are powerful systems for high-level expression of recombinant proteins, but due to the lack of endogenous vitamin K-dependent carboxylase, expression of functional FVII using this system is impossible. In the present study, we report a simple but versatile method to overcome the defect for high-level expression of the functional recombinant coagulation FVII in Sf9 cells. This method involves simultaneous expression of both human gamma-carboxylase (hGC) and human FVII genes in the host. It may be possible to express other vitamin K-dependent coagulation factors using this method in the future.

Gla-Rich Protein Is a Novel Vitamin K-Dependent Protein Present in Serum That Accumulates at Sites of Pathological Calcifications

Mineralization of soft tissues is an abnormal process that occurs in any body tissue and can greatly increase morbidity and mortality. Vitamin K-dependent (VKD) proteins play a crucial role in these processes; matrix Gla protein is considered one of the most relevant physiological inhibitors of soft tissue calcification know to date. Several studies have suggested that other, still unknown, VKD proteins might also be involved in soft tissue calcification pathologies. We have recently identified in sturgeon a new VKD protein, Gla-rich protein (GRP), which contains the highest ratio between number of Gla residues and size of the mature protein so far identified. Although mainly expressed in cartilaginous tissues of sturgeon, in rat GRP is present in both cartilage and bone. We now show that GRP is a circulating protein that is also expressed and accumulated in soft tissues of rats and humans, including the skin and vascular system in which, when affected by pathological calcifications, GRP accumulates at high levels at sites of mineral deposition, indicating an association with calcification processes. The high number of Gla residues and consequent mineral binding affinity properties strongly suggest that GRP may directly influence mineral formation, thereby playing a role in processes involving connective tissue mineralization.

Genetic mutation of vitamin K-dependent gamma-glutamyl carboxylase domain in patients with calcium oxalate urolithiasis

To investigate the exon mutation of vitamin K-dependent gamma-glutamyl carboxylase (GGCX or VKDC) in patients with calcium oxalate urolithasis, renal cortex and peripheral blood samples were obtained from severe hydronephrosis patients (with or without calculi), and renal tumor patients undergoing nephrectomy. GGCX mutations in all 15 exons were examined in 44 patients with calcium oxalate urolithiasis (COU) by polymerase chain reaction (PCR) and denatured high pressure liquid chromatography (DHPLC), and confirmed by sequencing. Mutation was not found in all COU samples compared to the controls. These data demonstrated that functional GGCX mutations in all 15 exons do not occur in most COU patients. It was suggested that there may be no significant association between the low activity and mutation of GGCX in COU.

Vitamin K-dependent proteins in the developing and aging nervous system.

Although the warfarin embryopathy syndrome, with its neurologic and bone abnormalities, has been known for decades, the role of vitamin K in the brain has not been studied systematically. Recently, it was demonstrated that vitamin K-dependent carboxylase expression is temporally regulated in a tissue-specific manner with high expression in the nervous system during the early embryonic stages and with liver expression after birth and in adult animals. This finding, along with the discovery of wide distribution of the novel vitamin K-dependent growth factor, Gas6, in the central nervous system, provides compelling evidence of a biologic role of vitamin K during the development of the nervous system. In animals and bacteria, vitamin K was observed to influence the brain sulfatide concentration and the activity and synthesis of an important enzyme involved in brain sphingolipids biosynthesis. Taken together, previous research results point to a possible role of vitamin K in the nervous system, especially during its development. Hence, the knowledge of the biologic role of vitamin K in the brain may be important for unveiling the mechanisms of normal and pathologic development and aging of the nervous system. The role of the vitamin K-dependent protein Gas6 in activation of signal transduction events in the brain in light of the age-related changes in the nervous system is also discussed.

VITAMIN K-DEPENDENT CARBOXYLASE IN SKIN

Journal Article Vitamin K-Dependent Carboxylase in Skin Get access Nutrition Reviews, Volume 45, Issue 8, August 1987, Pages 190–192, https://doi.org/10.1111/j.1753-4887.1987.tb06086.x Published: 01 August 1987

Insight into the Coupling Mechanism of the Vitamin K-Dependent Carboxylase: Mutation of Histidine 160 Disrupts Glutamic Acid Carbanion Formation and Efficient Coupling of Vitamin K Epoxidation to Glutamic Acid Carboxylation

Vitamin K-dependent (VKD) proteins become activated by the VKD carboxylase, which converts Glu's to carboxylated Glu's (Gla's) in their Gla domains. The carboxylase uses vitamin K epoxidation to drive Glu carboxylation, and the two half-reactions are coupled in 1:1 stoichiometry by an unknown mechanism. We now report the first identification of a residue, His160, required for coupling. A H160A mutant showed wild-type levels of epoxidation but substantially less carboxylation. Monitoring proton abstraction using a peptide with Glu tritiated at the gamma-carbon position revealed that poor coupling was due to impaired carbanion formation. H160A showed a 10-fold lower ratio of tritium release to vitamin K epoxidation than wild-type enzyme (i.e., 0.12 versus 1.14, respectively), which could fully account for the fold decrease in coupling efficiency. The Ala substitution in His160 did not affect the K m for vitamin K and caused only a 2-fold increase in the K m for Glu and 2-fold decrease in the activation of vitamin K epoxidation by Glu. The H160A K m for CO 2 was 5-fold higher than the wild-type enzyme. However, the k cat for H160A carboxylation was 8-9-fold lower than the wild-type enzyme with all three substrates (i.e., Glu, CO 2, and vitamin K), suggesting a catalytic role for His160 in carbanion formation. We propose that His160 facilitates the formation of the transition state for carbanion formation. His160 is highly conserved in metazoan VKD carboxylases but not in some bacterial orthologues (acquired by horizontal gene transfer), which has implications for how bacteria have adapted the carboxylase for novel functions.

Vitamin K Induces Osteoblast Differentiation through Pregnane X Receptor-Mediated Transcriptional Control of the Msx2 Gene

Vitamin K is a fat-soluble vitamin that serves as a coenzyme for vitamin K-dependent carboxylase. Besides its canonical action, vitamin K binds to the steroid and xenobiotic receptor (SXR)/pregnane X receptor (PXR) and modulates gene transcription. To determine if the osteoprotective action of vitamin K is the result of the PXR/SXR pathway, we screened by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis the PXR/SXR target genes in an osteoblastic cell line (MC3T3-E1) treated with a vitamin K2 (menaquinone 4 [MK4]). Osteoblastic differentiation of MC3T3-E1 cells was induced by MK4. Msx2, an osteoblastogenic transcription factor, was identified as an MK4-induced gene. Functional analysis of the Msx2 gene promoter mapped a vitamin K-responsive element (PXR-responsive element [PXRE]) that was directly bound by a PXR/retinoid X receptor alpha heterodimer. In a chromatin immunoprecipitation analysis, PXR was recruited together with a coactivator, p300, to the PXRE in the Msx2 promoter. MK4-bound PXR cooperated with estrogen-bound estrogen receptor alpha to control transcription at the Msx2 promoter. Knockdown of either PXR or Msx2 attenuated the effect of MK4 on osteoblastic differentiation. Thus, the present study suggests that Msx2 is a target gene for PXR activated by vitamin K and suggests that the osteoprotective action of MK4 in the human mediates, at least in part, a genomic pathway of vitamin K signaling.

Expression of OPN in rat kidney with different doses of vitamin K3 injection

Osteopontin (OPN) is a phosphorylated glycoprotein that occurs in high levels in bone, but is also present at sites of pathological calcification. Based on autotitration, OPN is regarded as a potent inhibitor of hydroxyapatite crystal formation, an activity that requires phosphorylation of the protein. Vitamin K is also a stone inhibitor, which has an effect on vitamin K-dependent gamma-glutamyl carboxylase activity. To explore the mechanism, using different doses of vitamin-K, we developed an enzyme-linked immunosorbent assay (ELISA) using a combination of mouse monoclonal antibodies against OPN in the rat kidney. We also investigated the expression of OPN mRNA in the kidney in an experimental model of a renal stone, using mass spectrometry to identify 27 serines and two threonines that are phosphorylated in the stone isoform of OPN.

Expression of a Two Chain Gamma-Glutamyl Carboxylase: Importance of Disulfide Bond Formation and Transmembrane Domain Interactions.

The vitamin K-dependent carboxylase is an integral membrane protein with five transmembrane domains (TMDs). It catalyzes the post-translational modification of specific glutamic acid residues of vitamin K-dependent proteins to gamma-carboxyglutamic acid residues. This posttranslational modification is critical for the biological functions of blood coagulation. The native enzyme is a single chain molecule with one disulfide bond. In this study, we have expressed carboxylase as two chains: residues 1–345 and 346–758 in the same insect cells. Our results show that these two fragments are assembled into a fully active enzyme and are joined by a disulfide. Affinity purification of the carboxylase C-terminal fragment (346–758) results in co-purification of the N-terminal fragment (1–345) even under reducing condition. This indicates that, in addition to the disulfide linkage between these two fragments, they are also linked by non-covalent interactions. One possibility is that the hydrophobic interactions between the TMDs play a role. According to carboxylase membrane topology, there are four TMDs (1–4) in the N-terminal fragment and one TMD (fifth) in the C-terminal fragment. The C-terminal fragment contains all glycosylation sites. When we introduced two prolines to disrupt the transmembrane helix in the wild type carboxylase's fifth TMD, glycosylation was eliminated. This indicates that the domain is not inserted into the lumen of the ER, but remains in the cytoplasm. Therefore, as our results demonstrate, in the two chain carboxylase with its fifth TMD disrupted, the two chains do not form a disulfide bound nor do they associate through essential non-covalent TMD interactions. While proline residues can disrupt membrane helices as described above, they often occur at the interface between the membrane and the lumenal surface of ER; these prolines appear to affect the chain orientation as it exits the membrane. There is a proline at residue 378 near the lumenal surface of the fifth TMD helix of carboxylase. To examine P378's effect on disulfide bond formation, we mutated it to leucine. Results show that less disulfide bond formed in the two chain mutant carboxylase and the protein was significantly degraded when compared to the unmutated two chain molecule. Based on our results, we conclude the following:1) the two chain carboxylase is assembled into a single molecule in vivo and the two chains are joined by a disulfide bond, the enzyme carboxylates gla-containing substrates and binds propeptide with affinity similar to that of wild type enzyme. Therefore, this molecule is a good model for structural studies of TMD interactions and disulfide bond formation;2) TMD association in the membrane is important for the orientation of the N- and C-terminal portions of carboxylase to be assembled into the active enzyme;3) and finally proline residue 378 at the lumenal interface of the fifth TMD plays a key role in the conformation which promotes disulfide formation.

Identification of the N-Linked Glycosylation Sites of Vitamin K-Dependent Carboxylase and Effect of Glycosylation on Carboxylase Function

The vitamin K-dependent carboxylase is an integral membrane protein which is required for the post-translational modification of a variety of vitamin K-dependent proteins. Previous studies have suggested carboxylase is a glycoprotein with N-linked glycosylation sites. In this study, we identify the N-glycosylation sites of carboxylase by mass spectrometric peptide mapping analyses combined with site-directed mutagenesis. Our mass spectrometric results show that the N-linked glycosylation in carboxylase occurs at positions N459, N550, N605, and N627. Eliminating these glycosylation sites by changing asparagine to glutamine caused the mutant carboxylase to migrate faster on SDS-PAGE gels, adding further evidence that these sites are glycosylated. In addition, the mutation studies identified N525, a site that cannot be recovered by mass spectroscopy analysis, as a glycosylation site. Furthermore, the potential glycosylation site at N570 is glycosylated only if all five natural glycosylation sites are simultaneously mutated. Removal of the oligosaccharides by glycosidase from wild-type carboxylase or by elimination of the functional glycosylation sites by site-directed mutagenesis did not affect either the carboxylation or epoxidation activity when the small FLEEL pentapeptide was used as a substrate, suggesting that N-linked glycosylation is not required for the enzymatic function of carboxylase. In contrast, when site N570 and the five natural glycosylation sites were mutated simultaneously, the resulting carboxylase protein was degraded. Our results suggest that N-linked glycosylation is not essential for carboxylase enzymatic activity but is important for protein folding and stability.

Brønsted Analysis Reveals Lys218 as the Carboxylase Active Site Base That Deprotonates Vitamin K Hydroquinone To Initiate Vitamin K-Dependent Protein Carboxylation

The vitamin K-dependent (VKD) carboxylase converts Glu's to carboxylated Glu's in VKD proteins to render them functional in a broad range of physiologies. The carboxylase uses vitamin K hydroquinone (KH(2)) epoxidation to drive Glu carboxylation, and one of its critical roles is to provide a catalytic base that deprotonates KH(2) to allow epoxidation. A long-standing model invoked Cys as the catalytic base but was ruled out by activity retention in a mutant where every Cys is substituted by Ala. Inhibitor analysis of the cysteine-less mutant suggested that the base is an activated amine [Rishavy et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 13732-13737], and in the present study, we used an evolutionary approach to identify candidate amines, which revealed His160, His287, His381, and Lys218. When mutational analysis was performed using an expression system lacking endogenous carboxylase, the His to Ala mutants all showed full epoxidase activity but K218A activity was not detectable. The addition of exogenous amines restored K218A activity while having little effect on wild type carboxylase, and pH studies indicated that rescue was dependent upon the basic form of the amine. Importantly, Brønsted analysis that measured the effect of amines with different pK(a) values showed that K218A activity rescue depended upon the basicity of the amine. The combined results provide strong evidence that Lys218 is the essential base that deprotonates KH(2) to initiate the reaction. The identification of this base is an important advance in defining the carboxylase active site and has implications regarding carboxylase membrane topology and the feedback mechanism by which the Glu substrate regulates KH(2) oxygenation.

Novel γ‐carboxyglutamic acid‐containing peptides from the venom of Conus textile

The cone snail is the only invertebrate system in which the vitamin K-dependent carboxylase (or gamma-carboxylase) and its product gamma-carboxyglutamic acid (Gla) have been identified. It remains the sole source of structural information of invertebrate gamma-carboxylase substrates. Four novel Gla-containing peptides were purified from the venom of Conus textile and characterized using biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have six Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino acid 6-l-bromotryptophan. Gla(2)-TxVI/A and Gla(2)-TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids -46 to -27) were nearly identical and highly hydrophobic. The predicted propeptide region (-20 to -1) that contains the gamma-carboxylation recognition site (gamma-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conusgamma-carboxylase and synthetic peptide substrates localized the gamma-CRS of Gla(1)-TxVI to the region -14 to -1 of the polypeptide precursor: the Km was reduced from 1.8 mm for Gla (1)-TxVI lacking a propeptide to 24 microm when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km value tenfold.

279 Novel splicing variant of the vitamin k-dependent γ-glutamyl carboxylase (GGCX) causes des-gamma-carboxyl prothrombin production in human hepatocellular carcinoma cell lines

279 Novel splicing variant of the vitamin k-dependent γ-glutamyl carboxylase (GGCX) causes des-gamma-carboxyl prothrombin production in human hepatocellular carcinoma cell lines

Identification of the N-Linked Glycosylation Sites of Vitamin K-Dependent Carboxylase and Effect of Glycosylation on Carboxylase Function †

Identification of the N-Linked Glycosylation Sites of Vitamin K-Dependent Carboxylase and Effect of Glycosylation on Carboxylase Function †