Distinct enzymatic strategies for de novo generation of disulfide bonds in membranes.

Disulfide bond formation is a catalyzed reaction essential for the folding and stability of proteins in the secretory pathway. In prokaryotes, disulfide bonds are generated by DsbB or VKOR homologs that couple the oxidation of a cysteine pair to quinone reduction. Vertebrate VKOR and VKOR-like enzymes have gained the epoxide reductase activity to support blood coagulation. The core structures of DsbB and VKOR variants share the architecture of a four-transmembrane-helix bundle that supports the coupled redox reaction and a flexible region containing another cysteine pair for electron transfer. Despite considerable similarities, recent high-resolution crystal structures of DsbB and VKOR variants reveal significant differences. DsbB activates the cysteine thiolate by a catalytic triad of polar residues, a reminiscent of classical cysteine/serine proteases. In contrast, bacterial VKOR homologs create a hydrophobic pocket to activate the cysteine thiolate. Vertebrate VKOR and VKOR-like maintain this hydrophobic pocket and further evolved two strong hydrogen bonds to stabilize the reaction intermediates and increase the quinone redox potential. These hydrogen bonds are critical to overcome the higher energy barrier required for epoxide reduction. The electron transfer process of DsbB and VKOR variants uses slow and fast pathways, but their relative contribution may be different in prokaryotic and eukaryotic cells. The quinone is a tightly bound cofactor in DsbB and bacterial VKOR homologs, whereas vertebrate VKOR variants use transient substrate binding to trigger the electron transfer in the slow pathway. Overall, the catalytic mechanisms of DsbB and VKOR variants have fundamental differences.

Warfarin analogs target disulfide bond-forming enzymes and suggest a residue important for quinone and coumarin binding.

Disulfide bond formation has a central role in protein folding of both eukaryotes and prokaryotes. In bacteria, disulfide bonds are catalyzed by DsbA and DsbB/VKOR enzymes. First, DsbA, a periplasmic disulfide oxidoreductase, introduces disulfide bonds into substrate proteins. Then, the membrane enzyme, either DsbB or VKOR, regenerate DsbA's activity by the formation of de novo disulfide bonds which reduce quinone. We have previously performed a high-throughput chemical screen and identified a family of warfarin analogs that target either bacterial DsbB or VKOR. In this work, we expressed functional human VKORc1 in Escherichia coli and performed a structure-activity-relationship analysis to study drug selectivity between bacterial and mammalian enzymes. We found that human VKORc1 can function in E. coli by removing two positive residues, allowing the search for novel anticoagulants using bacteria. We also found one warfarin analog capable of inhibiting both bacterial DsbB and VKOR and a second one antagonized only the mammalian enzymes when expressed in E. coli. The difference in the warfarin structure suggests that substituents at positions three and six in the coumarin ring can provide selectivity between the bacterial and mammalian enzymes. Finally, we identified the two amino acid residues responsible for drug binding. One of these is also essential for de novo disulfide bond formation in both DsbB and VKOR enzymes. Our studies highlight a conserved role of this residue in de novo disulfide-generating enzymes and enable the design of novel anticoagulants or antibacterials using coumarin as a scaffold.

Development of a sensor for disulfide bond formation in diverse bacteria.

In bacteria, disulfide bonds contribute to the folding and stability of proteins important for processes in the cellular envelope. In Escherichia coli, disulfide bond formation is catalyzed by DsbA and DsbB enzymes. DsbA is a periplasmic protein that catalyzes disulfide bond formation in substrate proteins, while DsbB is an inner membrane protein that transfers electrons from DsbA to quinones, thereby regenerating the DsbA active state. Actinobacteria including mycobacteria use an alternative enzyme named VKOR, which performs the same function as DsbB. Disulfide bond formation enzymes, DsbA and DsbB/VKOR, represent novel drug targets because their inhibition could simultaneously affect the folding of several cell envelope proteins including virulence factors, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. We have previously developed a cell-based and target-based assay to identify molecules that inhibit the DsbB and VKOR in pathogenic bacteria, using E. coli cells expressing a periplasmic ß-Galactosidase sensor (ß-Galdbs), which is only active when disulfide bond formation is inhibited. Here, we report the construction of plasmids that allows fine-tuning of the expression of the ß-Galdbs sensor and can be mobilized into other gram-negative organisms. As an example, when expressed in Pseudomonas aeruginosa UCBPP-PA14, which harbors two DsbB homologs, ß-Galdbs behaves similarly as in E. coli, and the biosensor responds to the inhibition of the two DsbB proteins. Thus, these ß-Galdbs reporter plasmids provide a basis to identify novel inhibitors of DsbA and DsbB/VKOR in multidrug-resistant gram-negative pathogens and to further study oxidative protein folding in diverse gram-negative bacteria. IMPORTANCE: Disulfide bonds contribute to the folding and stability of proteins in the bacterial cell envelope. Disulfide bond-forming enzymes represent new drug targets against multidrug-resistant bacteria because inactivation of this process would simultaneously affect several proteins in the cell envelope, including virulence factors, toxins, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. Identifying the enzymes involved in disulfide bond formation in gram-negative pathogens as well as their inhibitors can contribute to the much-needed antibacterial innovation. In this work, we developed sensors of disulfide bond formation for gram-negative bacteria. These tools will enable the study of disulfide bond formation and the identification of inhibitors for this crucial process in diverse gram-negative pathogens.

Distribution of non-synonymous Vkorc1 mutations in roof rats (Rattus rattus) in France and in Spain - consequences for management.

Rodent control is mainly done using anticoagulant rodenticides leading to the death of rodents through internal bleeding by targeting the VKORC1 protein. However, mutations in VKORC1 can lead to resistance to anticoagulant rodenticides that can cause treatment failure in the field. This study provides the first insight into the distribution, frequency and characterization of Vkorc1 mutations in roof rats (Rattus rattus) in France and in three administrative areas of Spain. The roof rat is present in France while it was thought to have almost disappeared with the expansion of the brown rat. Nevertheless, it has been found mainly in maritime areas. 151 roof rats out of 219 tested presented at least one missense mutation in the coding sequences of Vkorc1 gene (i.e. 69.0% of the rat). Nine Vkorc1 genotypes were detected (Y25F, A26P, R40G, S57F, W59C, W59R, H68N, Y25F/K152T and Y25F/W59R. Biochemical characterization of the consequences of these different genotypes proved that these various genotypes did not induce severe resistance to anticoagulant rodenticides. Even if many mutations of the Vkorc1 gene are present in roof rat populations in France, their management may be based in a first approach, considering the low levels of resistance induced, on the use of first-generation anticoagulants less dangerous for wildlife. The use of second-generation may be considered when treatment failure is observed or when bait consumption is limited.

Determining the necessity of phenyl ring π-character in warfarin.

Despite the difficulty in administering a safe dose regimen and reports of emerging resistance, warfarin (1) remains the most widely-used oral anticoagulant for the prevention and treatment of thrombosis in humans globally. Systematic substitution of the warfarin phenyl ring with either 1,3,5,7-cyclooctatetraene (COT) (2), cubane (3), cyclohexane (4) or cyclooctane (5) and subsequent evaluation against the target enzyme, vitamin K epoxide reductase (VKOR), facilitated interrogation of both steric and electronic properties of the phenyl pharmacophore. The tolerance of VKOR to further functional group modification (carboxylate 14, PTAD adduct 15) was also investigated. The results demonstrate the importance of both annulene conferred π-interactions and ring size in the activity of warfarin.

Identification of the Thioredoxin Partner of Vitamin K Epoxide Reductase in Mycobacterial Disulfide Bond Formation.

Disulfide bonds influence the stability and activity of many proteins. In Escherichia coli, the DsbA and DsbB enzymes promote disulfide bond formation. Other bacteria, including the Actinobacteria, use instead of DsbB the enzyme vitamin K epoxide reductase (VKOR), whose gene is found either fused to or in the same operon as a dsbA-like gene. Mycobacterium tuberculosis and other Gram-positive actinobacteria secrete many proteins with even numbers of cysteines to the cell envelope. These organisms have predicted oxidoreductases and VKOR orthologs. These findings indicate that such bacteria likely form disulfide bonds in the cell envelope. The M. tuberculosisvkor gene complements an E. colidsbB deletion strain, restoring the oxidation of E. coli DsbA. While we have suggested that the dsbA gene linked to the vkor gene may express VKOR's partner in mycobacteria, others have suggested that two other extracytoplasmic oxidoreductases (DsbE or DsbF) may be catalysts of protein disulfide bond formation. However, there is no direct evidence for interactions of VKOR with either DsbA, DsbE, or DsbF. To identify the actual substrate of VKOR, we identified two additional predicted extracytoplasmic DsbA-like proteins using bioinformatics analysis of the M. tuberculosis genome. Using the five potential DsbAs, we attempted to reconstitute disulfide bond pathways in E. coli and in Mycobacterium smegmatis, a close relative of M. tuberculosis Our results show that only M. tuberculosis DsbA is oxidized by VKOR. Comparison of the properties of dsbA- and vkor-null mutants in M. smegmatis shows parallels to the properties of dsb mutations in E. coliIMPORTANCE Disulfide bond formation has a great impact on bacterial pathogenicity. Thus, disulfide-bond-forming proteins represent new targets for the development of antibacterials, since the inhibition of disulfide bond formation would result in the simultaneous loss of the activity of several classes of virulence factors. Here, we identified five candidate proteins encoded by the M. tuberculosis genome as possible substrates of the M. tuberculosis VKOR protein involved in disulfide bond formation. We then reconstituted the mycobacterial disulfide bond formation pathway in E. coli and showed that of the five candidates, only M. tuberculosis DsbA is efficiently oxidized by VKOR in E. coli We also present evidence for the involvement of VKOR in DsbA oxidation in M. smegmatis.

Aeropyrum pernix membrane topology of protein VKOR promotes protein disulfide bond formation in two subcellular compartments.

Disulfide bonds confer stability and activity to proteins. Bioinformatic approaches allow predictions of which organisms make protein disulfide bonds and in which subcellular compartments disulfide bond formation takes place. Such an analysis, along with biochemical and protein structural data, suggests that many of the extremophile Crenarachaea make protein disulfide bonds in both the cytoplasm and the cell envelope. We have sought to determine the oxidative folding pathways in the sequenced genomes of the Crenarchaea, by seeking homologues of the enzymes known to be involved in disulfide bond formation in bacteria. Some Crenarchaea have two homologues of the cytoplasmic membrane protein VKOR, a protein required in many bacteria for the oxidation of bacterial DsbAs. We show that the two VKORs of Aeropyrum pernix assume opposite orientations in the cytoplasmic membrane, when expressed in E. coli. One has its active cysteines oriented toward the E. coli periplasm (ApVKORo) and the other toward the cytoplasm (ApVKORi). Furthermore, the ApVKORo promotes disulfide bond formation in the E. coli cell envelope, while the ApVKORi promotes disulfide bond formation in the E. coli cytoplasm via a co-expressed archaeal protein ApPDO. Amongst the VKORs from different archaeal species, the pairs of VKORs in each species are much more closely related to each other than to the VKORs of the other species. The results suggest two independent occurrences of the evolution of the two topologically inverted VKORs in archaea. Our results suggest a mechanistic basis for the formation of disulfide bonds in the cytoplasm of Crenarchaea.

Disulfide bond formation in prokaryotes: history, diversity and design.

The formation of structural disulfide bonds is essential for the function and stability of a great number of proteins, particularly those that are secreted. There exists a variety of dedicated cellular catalysts and pathways from archaea to humans that ensure the formation of native disulfide bonds. In this review we describe the initial discoveries of these pathways and report progress in recent years in our understanding of the diversity of these pathways in prokaryotes, including those newly discovered in some archaea. We will also discuss the various successful efforts to achieve laboratory-based evolution and design of synthetic disulfide bond formation machineries in the bacterium Escherichia coli. These latter studies have also led to new more general insights into the redox environment of the cytoplasm and bacterial cell envelope. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.

Tris(3-hydroxypropyl)phosphine is superior to dithiothreitol for in vitro assessment of vitamin K 2,3-epoxide reductase activity.

Use of the reductant dithiothreitol (DTT) as a substrate for measuring vitamin K 2,3-epoxide reductase (VKOR) activity in vitro has been reported to be problematic because it enables side reactions involving the vitamin K1 2,3-epoxide (K1>O) substrate. Here we characterize specific problems when using DTT and show that tris(3-hydroxypropyl)phosphine (THPP) is a reliable alternative to DTT for in vitro assessment of VKOR enzymatic activity. In addition, the pH buffering compound imidazole was found to be problematic in enhancing DTT-dependent non-enzymatic side reactions. Using THPP and phosphate-based pH buffering, we measured apparent Michaelis-Menten constants of 1.20 µM for K1>O and 260 µM for the active neutral form of THPP. The Km value for K1>O is in agreement with the value that we previously obtained using DTT (1.24 µM). Using THPP, we successfully eliminated non-enzymatic production of 3-hydroxyvitamin K1 and its previously reported base-catalyzed conversion to K1, both of which were shown to occur when DTT and imidazole are used as the reductant and pH buffer, respectively, in the in vitro VKOR assay. Accordingly, substitution of THPP for DTT in the in vitro VKOR assay will ensure more accurate enzymatic measurements and assessment of warfarin and other 4-hydroxycoumarin inhibition constants.

Rv2969c, essential for optimal growth in Mycobacterium tuberculosis, is a DsbA-like enzyme that interacts with VKOR-derived peptides and has atypical features of DsbA-like disulfide oxidases.

The bacterial disulfide machinery is an attractive molecular target for developing new antibacterials because it is required for the production of multiple virulence factors. The archetypal disulfide oxidase proteins in Escherichia coli (Ec) are DsbA and DsbB, which together form a functional unit: DsbA introduces disulfides into folding proteins and DsbB reoxidizes DsbA to maintain it in the active form. In Mycobacterium tuberculosis (Mtb), no DsbB homologue is encoded but a functionally similar but structurally divergent protein, MtbVKOR, has been identified. Here, the Mtb protein Rv2969c is investigated and it is shown that it is the DsbA-like partner protein of MtbVKOR. It is found that it has the characteristic redox features of a DsbA-like protein: a highly acidic catalytic cysteine, a highly oxidizing potential and a destabilizing active-site disulfide bond. Rv2969c also has peptide-oxidizing activity and recognizes peptide segments derived from the periplasmic loops of MtbVKOR. Unlike the archetypal EcDsbA enzyme, Rv2969c has little or no activity in disulfide-reducing and disulfide-isomerase assays. The crystal structure of Rv2969c reveals a canonical DsbA fold comprising a thioredoxin domain with an embedded helical domain. However, Rv2969c diverges considerably from other DsbAs, including having an additional C-terminal helix (H8) that may restrain the mobility of the catalytic helix H1. The enzyme is also characterized by a very shallow hydrophobic binding surface and a negative electrostatic surface potential surrounding the catalytic cysteine. The structure of Rv2969c was also used to model the structure of a paralogous DsbA-like domain of the Ser/Thr protein kinase PknE. Together, these results show that Rv2969c is a DsbA-like protein with unique properties and a limited substrate-binding specificity.

In silico evaluation of pharmacokinetic parameters, delivery, distribution and anticoagulative effects of new 4,7-dihydroxycoumarin derivative.

In this study, pharmacological profiling and investigation of the anticoagulant activity of the newly synthesized coumarin derivative: (E)-3-(1-((4-hydroxy-3-methoxyphenyl)amino)ethylidene)-2,4-dioxochroman-7-yl acetate (L) were performed. The obtained results were compared with the parameters obtained for Warfarin (WF), which is a standard good oral anticoagulant. The estimated high binding affinity of L toward plasma proteins (PPS% value is > 90%) justifies the investigation of binding affinity and comparative analysis of L and WF to Human Serum Albumin (HSA) using the spectrofluorimetric method (296, 303 and 310 K) as well as molecular docking and molecular dynamics simulations. Compound L shows a very good binding affinity especially to the active site of WF (the active site I -subdomain IIA), quenching HSA fluorescence by a static process. Also, the finite element smeared model (Kojic Transport Model, KTM), which includes blood vessels and tissue, was implemented to compute the convective-diffusion transport of L and WF within the liver. Finally, compound L shows a high degree of inhibitory activity toward the VKOR receptor comparable to the inhibitory activity of WF. Stabilization and limited flexibility of amino acid residues in the active site of the VKOR after binding of L and WF indicates a very good inhibitory potential of compound L. The high affinity of the L for the VKOR enzyme (Vitamin K antagonist), as well as the structural similarity to commercial anticoagulants (WF), provide a basis for further studies and potential application in the treatment of venous thrombosis, pulmonary embolism and ischemic heart disease.Communicated by Ramaswamy H. Sarma.

Characterization of Warfarin Inhibition Kinetics Requires Stabilization of Intramembrane Vitamin K Epoxide Reductases.

Intramembrane enzymes are often difficult for biochemical characterization. Human vitamin K epoxide reductase (VKOR) is the target of warfarin. However, this intramembrane enzyme becomes insensitive to warfarin inhibition in vitro, preventing the characterization of inhibition kinetics for decades. Here we employ structural biology methods to identify stable VKOR and VKOR-like proteins and purify them to near homogeneity. We find that the key to maintain their warfarin sensitivity is to stabilize their native protein conformation in vitro. Reduced glutathione drastically increases the warfarin sensitivity of a VKOR-like protein from Takifugu rubripes, presumably through maintaining a disulfide-bonded conformation. Effective inhibition of human VKOR-like requires also the use of LMNG, a mild detergent developed for crystallography to increase membrane protein stability. Human VKOR needs to be preserved in ER-enriched microsomes to exhibit warfarin sensitivity, whereas human VKOR purified in LMNG is stable only with pre-bound warfarin. Under these optimal conditions, warfarin inhibits with tight-binding kinetics. Overall, our studies show that structural biology methods are ideal for stabilizing intramembrane enzymes. Optimizing toward their inhibitor-binding conformation enables the characterization of enzyme kinetics in difficult cases.

Comparison of two reducing agents dithiothreitol and tris(3-hydroxypropyl)phosphine for in vitro kinetic assay of vitamin K epoxide reductase.

Vitamin K epoxide reductase (VKOR) is a target enzyme for anticoagulants, such as warfarin, that are used as medicines or rodenticides. Assessing VKOR activity is required to ensure the proper usage of these drugs. Dithiothreitol (DTT) is a typical disulfide reductant that is used as a substrate for in vitro VKOR assays. However, DTT is considered problematic because of its side effects. Tris(3-hydroxypropyl)phosphine (THP) has been found to be a reliable alternative to DTT, as shown by kinetic analyses of the VKOR with them. THP showed significantly lower V max and Km values than those of DTT; however, there was no significant difference in their V max/Km and IC50 for warfarin.

Genetic Polymorphism of VKORC1-1639 in Children With Intracranial Hemorrhage Due to Vitamin K Deficiency.

Intracranial hemorrhage due to vitamin K deficiency is a serious disease that can lead to morbidity, mortality, and mental retardation. Our goal in this study is to determine the frequency of VKORC1-1639 G>A polymorphism in patients who have undergone intracranial hemorrhage due to vitamin K deficiency bleeding (VKDB). To study VKORC1-1639 G>A polymorphism, blood was drawn from patients (n = 51, age 8:0 ± 6:5 years) followed at the Pediatric Neurology and Hematology section, Faculty of Medicine, Erciyes University, between 1990 and 2016, diagnosed with VKDB as idiopathic or from patients diagnosed with intracranial hemorrhage due to secondary vitamin K deficiency and also from volunteers (n = 51, age 11 ± 4.5 years). Intensive care and nutrition needs of patients and the laboratory radiological imaging results and treatments that were applied were analyzed through scanning the files of the patients and information received from families. Through detailed physical examination, patients with neurologic sequelae and ongoing epilepsy were determined. The results were compared to clinical and laboratory results with control group. Eight (15.7%) of the patients were normal, 29 (56.9%) heterozygous carrier, and 14 (27.5%) homozygous mutants. In the control group, 19 (37.3%) were normal, 19 (37.3%) heterozygous carriers, and 13 (25.5%) homozygous mutants. The VKOR1-1639>A (SNP:rs9923231) mutant positivity (homozygous plus heterozygous mutant) was significantly higher in the patient group when compared to controls. There were no significant differences between patient and control groups in terms of the prognosis.

Dicoumarol: A Drug which Hits at Least Two Very Different Targets in Vitamin K Metabolism.

Dicoumarol, a symmetrical biscoumarin can be considered as the "parent" of the widely used anticoagulant drug, warfarin. The discovery of dicoumarol's bioactive properties resulted from an investigation into a mysterious cattle disease in the 1940s. It was then developed as a pharmaceutical, but was superseded in the 1950s by warfarin. Both dicoumarol and warfarin antagonise the blood clotting process through inhibition of vitamin K epoxide reductase (VKOR). This blocks the recycling of vitamin K and prevents the γ-carboxylation of glutamate residues in clotting factors. VKOR is an integral membrane protein and our understanding of the molecular mechanism of action of dicoumarol and warfarin is hampered by the lack of a three dimensional structure. There is consequent controversy about the membrane topology of VKOR, the location of the binding site for coumarin inhibitors and the mechanism of inhibition by these compounds. Dicoumarol (and warfarin) also inhibit a second enzyme, NAD(P)H quinone oxidoreductase 1 (NQO1). This soluble, cytoplasmic enzyme may also play a minor role in the recycling of vitamin K. However, its main cellular roles as an enzyme appear to be detoxification and the prevention of the build-up of reactive oxygen species. NQO1 is well characterised biochemically and structurally. Consequently, structure-based drug design has identified NQO1 inhibitors which have potential for the development of anti-cancer drugs. Many of these compounds are structurally related to dicoumarol and some have reduced "off target" effects. Therefore, it is possible that dicoumarol will become the "parent" of a second group of drugs.

Pharmacological effects of a vitamin K1 2,3-epoxide reductase (VKOR) inhibitor, 3-acetyl-5-methyltetronic acid, on cisplatin-induced fibrosis in rats.

Cisplatin (CDDP) is a chemotherapeutic agent that is widely used in the treatment of lymphomas and solid malignancies. However, its clinical usage is limited by its severe side effects in the kidneys. Glomerular and tubular injuries in the kidneys commonly progress to interstitial fibrosis and, ultimately, the end stage of renal failure. We previously reported that 3-acetyl-5-methyltetronic acid (AMT) had inhibitory effects on rat renal vitamin K1 2,3-epoxide reductase (VKOR) in vitro and also suppressed mesangial cell proliferation and, consequently, the formation of fibrosis via the vitamin K-dependent activation of the growth arrest-specific 6 (Gas6)/Axl pathway in anti-Thy-1 glomerulonephritis (Thy-1 GN) in rats. In the present study, we demonstrated that AMT alleviated the progression of renal fibrosis in CDDP-treated rats. The repeated intravenous administration of AMT for 28 days dose-dependently suppressed increases in plasma urea nitrogen and plasma creatinine levels as well as creatinine clearance in CDDP-treated rats. Furthermore, the treatment suppressed the expression of α-smooth muscle actin (SMA)-positive cells and ameliorated the extracellular matrix accumulation of collagen III, indicating an antifibrotic effect. In conclusion, our toxicological and histopathological results demonstrated quantitatively the pharmacological inhibitory effects of AMT on the progression of renal fibrosis in CDDP-treated rats.

VKCFD2 - from clinical phenotype to molecular mechanism.

Vitamin K 2,3-epoxide reductase complex, subunit 1 (VKORC1) is an enzyme essential for the vitamin K cycle. VKORC1 catalyses the reduction of vitamin K 2,3-epoxide to the quinone form of vitamin K and further to vitamin K hydroquinone. The generated vitamin K hydroquinone serves as substrate for the enzyme γ-glutamyl-carboxylase which modifies all vitamin K-dependent proteins, allowing them to bind calcium ions necessary for physiological activity. Vitamin K-dependent proteins include the coagulation factors FII, FVII, FIX, FX, and proteins C, S und Z. Insufficient VKORC1 enzyme activity results in deficiency of the vitamin K-dependent clotting factors leading to haemorrhagic disorders. This phenotype is known as vitamin K clotting factor deficiency type 2 (VKCFD2). Worldwide, only four families of independent origin have been reported with this rare bleeding disorder. Affected family members carry the mutation VKORC1:p.Arg98Trp in homozygous form, the only mutation found so far to be associated with VKCFD2. Now, ten years after the identification of the VKORC1 gene, the molecular pathomechanism of VKCFD2 has been clarified. The Arg98Trp mutation disrupts an ER retention motif of VKORC1 leading to mislocalisation of the protein to outside the endoplasmatic reticulum. In this review, we summarize the clinical data, diagnosis, therapy and molecular pathomechanism of VKCFD2.

VKORC1 and VKORC1L1: Why do Vertebrates Have Two Vitamin K 2,3-Epoxide Reductases?

Among all cellular life on earth, with the exception of yeasts, fungi, and some prokaryotes, VKOR family homologs are ubiquitously encoded in nuclear genomes, suggesting ancient and important biological roles for these enzymes. Despite single gene and whole genome duplications on the largest evolutionary timescales, and the fact that most gene duplications eventually result in loss of one copy, it is surprising that all jawed vertebrates (gnathostomes) have retained two paralogous VKOR genes. Both VKOR paralogs function as entry points for nutritionally acquired and recycled K vitamers in the vitamin K cycle. Here we present phylogenetic evidence that the human paralogs likely arose earlier than gnathostomes, possibly in the ancestor of crown chordates. We ask why gnathostomes have maintained these paralogs throughout evolution and present a current summary of what we know. In particular, we look to published studies about tissue- and developmental stage-specific expression, enzymatic function, phylogeny, biological roles and associated pathways that together suggest subfunctionalization as a major influence in evolutionary fixation of both paralogs. Additionally, we investigate on what evolutionary timescale the paralogs arose and under what circumstances in order to gain insight into the biological raison d'être for both VKOR paralogs in gnathostomes.

Structural Modeling Insights into Human VKORC1 Phenotypes.

Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) catalyses the reduction of vitamin K and its 2,3-epoxide essential to sustain γ-carboxylation of vitamin K-dependent proteins. Two different phenotypes are associated with mutations in human VKORC1. The majority of mutations cause resistance to 4-hydroxycoumarin- and indandione-based vitamin K antagonists (VKA) used in the prevention and therapy of thromboembolism. Patients with these mutations require greater doses of VKA for stable anticoagulation than patients without mutations. The second phenotype, a very rare autosomal-recessive bleeding disorder caused by combined deficiency of vitamin K dependent clotting factors type 2 (VKCFD2) arises from a homozygous Arg98Trp mutation. The bleeding phenotype can be corrected by vitamin K administration. Here, we summarize published experimental data and in silico modeling results in order to rationalize the mechanisms of VKA resistance and VKCFD2.

Phylogeny of the Vitamin K 2,3-Epoxide Reductase (VKOR) Family and Evolutionary Relationship to the Disulfide Bond Formation Protein B (DsbB) Family.

In humans and other vertebrate animals, vitamin K 2,3-epoxide reductase (VKOR) family enzymes are the gatekeepers between nutritionally acquired K vitamins and the vitamin K cycle responsible for posttranslational modifications that confer biological activity upon vitamin K-dependent proteins with crucial roles in hemostasis, bone development and homeostasis, hormonal carbohydrate regulation and fertility. We report a phylogenetic analysis of the VKOR family that identifies five major clades. Combined phylogenetic and site-specific conservation analyses point to clade-specific similarities and differences in structure and function. We discovered a single-site determinant uniquely identifying VKOR homologs belonging to human pathogenic, obligate intracellular prokaryotes and protists. Building on previous work by Sevier et al. (Protein Science 14:1630), we analyzed structural data from both VKOR and prokaryotic disulfide bond formation protein B (DsbB) families and hypothesize an ancient evolutionary relationship between the two families where one family arose from the other through a gene duplication/deletion event. This has resulted in circular permutation of primary sequence threading through the four-helical bundle protein folds of both families. This is the first report of circular permutation relating distant a-helical membrane protein sequences and folds. In conclusion, we suggest a chronology for the evolution of the five extant VKOR clades.