Vitamin K2 Biosynthetic Enzyme,UBIAD1 Is Essential for Embryonic Development of Mice

UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K₂ biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1 ^−/−) mouse embryos failed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1 ^−/− embryonic stem (ES) cells failed to synthesize vitamin K₂ but were able to synthesize CoQ9, similar to wild-type ES cells. Ubiad1 ^+/− mice developed normally, exhibiting normal growth and fertility. Vitamin K₂ tissue levels and synthesis activity were approximately half of those in the wild-type, whereas CoQ9 tissue levels and synthesis activity were similar to those in the wild-type. Similarly, UBIAD1 expression and vitamin K₂ synthesis activity of mouse embryonic fibroblasts prepared from Ubiad1 ^+/− E15.5 embryos were approximately half of those in the wild-type, whereas CoQ9 levels and synthesis activity were similar to those in the wild-type. Ubiad1 ^−/− mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant Ubiad1 ^+/− mice. These results suggest that UBIAD1 is responsible for vitamin K₂ synthesis but may not be responsible for CoQ9 synthesis in mice. We propose that UBIAD1 plays a pivotal role in embryonic development by synthesizing vitamin K₂, but may have additional functions beyond the biosynthesis of vitamin K₂.     

Characterization of the Enzyme CbiH60 Involved in Anaerobic Ring Contraction of the Cobalamin (Vitamin B12) Biosynthetic Pathway

The anaerobic pathway for the biosynthesis of cobalamin (vitamin B₁₂) has remained poorly characterized because of the sensitivity of the pathway intermediates to oxygen and the low activity of enzymes. One of the major bottlenecks in the anaerobic pathway is the ring contraction step, which has not been observed previously with a purified enzyme system. The Gram-positive aerobic bacterium Bacillus megaterium has a complete anaerobic pathway that contains an unusual ring contraction enzyme, CbiH₆₀, that harbors a C-terminal extension with sequence similarity to the nitrite/sulfite reductase family. To improve solubility, the enzyme was homologously produced in the host B. megaterium DSM319. CbiH₆₀ was characterized by electron paramagnetic resonance and shown to contain a [4Fe-4S] center. Assays with purified recombinant CbiH₆₀ demonstrate that the enzyme converts both cobalt-precorrin-3 and cobalt factor III into the ring-contracted product cobalt-precorrin-4 in high yields, with the latter transformation dependent upon DTT and an intact Fe-S center. Furthermore, the ring contraction process was shown not to involve a change in the oxidation state of the central cobalt ion of the macrocycle.     

Biosynthetic Precursors of Vitamin K as Growth Promoters for Bacteroides melaninogenicus

The growth of a vitamin K-requiring strain of Bacteroides melaninogenicus was promoted by some postulated and proven biosynthetic precursors of bacterial menaquinones, 1,4-dihydroxy-2-naphthoic acid, shikimic acid, chorismic acid, and 4(2'-carboxyphenyl)-4-oxobutyric acid. Growth of the organism with [2',4-(14)C(2)]-4(2'-carboxy phenyl)-4-oxobutyric acid as the vitamin K replacement gave rise to a mixture of radioactive menaquinone-9 and menaquinone-10; the dilution factor for this incorporation was 1.8.     

Biosynthetic Dihydroorotate Dehydrogenase from Lactobacillus bulgaricus: Partial Characterization of the Enzyme

Some of the catalytic properties of the biosynthetic dihydroorotate dehydrogenase purified from an anaerobic bacterium, Lactobacillus bulgaricus, are described. Studies with p-hydroxymercuribenzoate, N-ethylmaleimide, and mercuric chloride showed that sulfhydryl groups are necessary for transfer of electrons from dihydroorotate to a variety of electron acceptors. Protection studies with substrates for the enzyme indicated that free sulfhydryl groups at or near the active center are required for catalytic activity. Evidence is presented for the production of superoxide free radicals during reaction of the enzyme with molecular oxygen. Inhibitor studies with Tiron indicated that reduction of cytochrome c by the enzyme may involve the superoxide free radical as an intermediate. Orotate, one of the substrates for the enzyme, has been found to be a competitive inhibitor for the dihydroorotate site. The K(i) for orotate as estimated by several techniques is 0.1 mM. The K(m) for dihydroorotate with ferricyanide as the electron acceptor is estimated to be 0.5 mM.     

纳豆芽孢杆菌发酵生产维生素K2的生物合成途径及其基因调控

阐述了用经过物理和化学诱变后,筛选得到的纳豆芽孢杆菌发酵生产维生素K2的方法,VK2的生物合成途径以及分子生物学机制,并简要介绍了VK2的临床应用。为进一步研究与开发VK2提供理论依据。     

Overproduction of a Functional Fatty Acid Biosynthetic Enzyme Blocks Fatty Acid Synthesis in Escherichia coli

β-Ketoacyl-acyl carrier protein (ACP) synthetase II (KAS II) is one of three Escherichia coli isozymes that catalyze the elongation of growing fatty acid chains by condensation of acyl-ACP with malonyl-ACP. Overexpression of this enzyme has been found to be extremely toxic to E. coli, much more so than overproduction of either of the other KAS isozymes, KAS I or KAS III. The immediate effect of KAS II overproduction is the cessation of phospholipid synthesis, and this inhibition is specifically due to the blockage of fatty acid synthesis. To determine the cause of this inhibition, we examined the intracellular pools of ACP, coenzyme A (CoA), and their acyl thioesters. Although no significant changes were detected in the acyl-ACP pools, the CoA pools were dramatically altered by KAS II overproduction. Malonyl-CoA increased to about 40% of the total cellular CoA pool upon KAS II overproduction from a steady-state level of around 0.5% in the absence of KAS II overproduction. This finding indicated that the conversion of malonyl-CoA to fatty acids had been blocked and could be explained if either the conversion of malonyl-CoA to malonyl-ACP and/or the elongation reactions of fatty acid synthesis had been blocked. Overproduction of malonyl-CoA:ACP transacylase, the enzyme catalyzing the conversion of malonyl-CoA to malonyl-ACP, partially relieved the toxicity of KAS II overproduction, consistent with a model in which high levels of KAS II blocks access of the other KAS isozymes to malonyl-CoA:ACP transacylase.     

Characterization of the CDP-2-Glycerol Biosynthetic Pathway in Streptococcus pneumoniae

Capsule polysaccharide (CPS) plays an important role in the virulence of Streptococcus pneumoniae and is usually used as the pneumococcal vaccine target. Glycerol-2-phosphate is found in the CPS of S. pneumoniae types 15A and 23F and is rarely found in the polysaccharides of other bacteria. The biosynthetic pathway of the nucleotide-activated form of glycerol-2-phosphate (NDP-2-glycerol) has never been identified. In this study, three genes (gtp1, gtp2, and gtp3) from S. pneumoniae 23F that have been proposed to be involved in the synthesis of NDP-2-glycerol were cloned and the enzyme products were expressed, purified, and assayed for their respective activities. Capillary electrophoresis was used to detect novel products from the enzyme-substrate reactions, and the structure of the product was elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Gtp1 was identified as a reductase that catalyzes the conversion of 1,3-dihydroxyacetone to glycerol, Gtp3 was identified as a glycerol-2-phosphotransferase that catalyzes the conversion of glycerol to glycerol-2-phosphate, and Gtp2 was identified as a cytidylyltransferase that transfers CTP to glycerol-2-phosphate to form CDP-2-glycerol as the final product. The kinetic parameters of Gtp1 and Gtp2 were characterized in depth, and the effects of temperature, pH, and cations on these two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-2-glycerol has been identified biochemically; this pathway provides a method to enzymatically synthesize this compound.Capsule polysaccharide (CPS) of Gram-positive bacteria, external to the cell wall, provides resistance to phagocytosis. CPS in Streptococcus pneumoniae is the most important virulence factor and the target of pneumococcal vaccines (2). Ninety individual CPS serotypes have been recognized so far by immunological and chemical techniques (9). Each has a structurally distinct CPS, composed of repeating oligosaccharide units joined by glycosidic linkages. The components of the repeat units are transferred from nucleoside diphosphate (NDP) derivatives. Among the 54 identified CPS structures, several sugars and related compounds have been found. Seven NDP-monosaccharide precursors (glucopyranose, N-acetylglucosamine, galactopyranose, N-acetylgalactosamine, 2-acetamido-4-amino-2,4,6-trideoxy-d-galactopyranose, ribitol-phosphate, and phosphorylcholine) are available from housekeeping metabolic pathways, and the biosynthetic genes for 14 NDP-monosaccharide precursors were found in the pneumococcal cps loci. Among the 14 components, the pathways of five (NDP-d-mannitol, NDP-d-arabinitol, NDP-ribofuranose [Rib], CDP-glycerol [CDP-Gro], and NDP-2-glycerol) are putative and have not yet been identified (1, 4).Glycerol-2-phosphate is rarely present in bacteria and has been found in S. pneumoniae types 15A and 23F. The NDP-2-glycerol biosynthetic pathway has been proposed to include three enzymes: Gtp1, Gtp2, and Gtp3. Gtp3 has been proposed to be a glyceraldehyde-2-phosphotransferase and to be involved in the synthesis of glyceraldehyde-2-phosphate from glyceraldehyde. Gtp1, a putative dehydrogenase, has been proposed to be responsible for the conversion of glyceraldehyde-2-phosphate to glycerol-2-phosphate. The last step of the synthesis of CDP-2-glycerol is catalyzed by the putative glycerol-2-phosphate cytidyltransferase Gtp2 (14). The three genes, gtp1, gtp2, and gtp3, have also been found to be present in the cps loci of S. pneumoniae serotypes 15B, 15C, 15F, 23A, 23B, 28A, and 28F (4). However, the biosynthetic pathway for NDP-2-glycerol has never been identified by molecular and biochemical methods.In this study, we found that the enzymes were not reactive by the previously proposed CDP-2-glycerol biosynthetic pathway. Therefore, a new pathway was proposed, and the three enzymes, Gtp1, Gtp2, and Gtp3, were identified and confirmed biochemically as 1,3-dihydroxyacetone/glyceraldehyde reductase, glycerol-2-phosphate cytidylyltransferase, and glycerol-2-phosphotransferase, respectively. This is the first report on the characterization of the CDP-2-glycerol biosynthetic pathway.     

Production and Characterization of a New Specific Antiserum Against the Taurine Putative Biosynthetic Enzyme Cysteine Sulfinate Decarboxylase

Abstract: We have shown previously that cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme of taurine in the brain, is identical to the liver enzyme according to biochemical, kinetic, and immunochemical criteria. In the present work, CSD was purified in its native form from rat liver. The purification was performed in eight steps, which included conventional chromatography (diethylaminoethyl cellulose, hydroxylapatite), followed by HPLC (hydrophobic, adsorption, and ion-exchange HPLC). The purification factor was 11,000, and the final yield was around 2%. The procedure led to the enrichment of a protein, the molecular mass of which was 51,000 daltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final fraction was more than 90% homogeneous. By using this fraction as the antigen, an antiserum was raised in rabbit that (a) quantitatively immunoprecipitated CSD activity from liver and brain extract, and (b) immunolabeled one band (51,000 daltons) on immunoblots of partially purified fractions from liver. Enrichment of CSD specific activity and that of the protein immunolabeled by the antiserum for a given step, e.g., hydrophobic HPLC, were consistently parallel. The antiserum was used to carry out CSD immunocytochemistry in cerebellum. Numerous small cells were labeled in the Purkinje cell layer, the granular layer, and the white matter. In the molecular layer, Bergmann radial fibers were im munostained. The Purkinje and stellate cells were devoid of any labeling at the cell body and terminal levels. The antiserum appears to be specific for CSD and suitable for immunocytochemical visualization of CSD in the brain.     

Active Site Binding and Catalytic Role of Bicarbonate in 1,4-Dihydroxy-2-naphthoyl Coenzyme A Synthases from Vitamin K Biosynthetic Pathways

1,4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase, or MenB, catalyzes a carbon-carbon bond formation reaction in the biosynthesis of both vitamin K1 and K2. Bicarbonate is crucial to the activity of a large subset of its orthologues but lacks a clearly defined structural and mechanistic role. Here we determine the crystal structure of the holoenzymes from Escherichia coli at 2.30 ? and Synechocystis sp. PCC6803 at 2.04 ?, in which the bicarbonate cofactor is bound to the enzyme active site at a position equivalent to that of the side chain carboxylate of an aspartate residue conserved among bicarbonate-insensitive DHNA-CoA synthases. Binding of the planar anion involves both nonspecific electrostatic attraction and specific hydrogen bonding and hydrophobic interactions. In the absence of bicarbonate, the anion binding site is occupied by a chloride ion or nitrate, an inhibitor directly competing with bicarbonate. These results provide a solid structural basis for the bicarbonate dependence of the enzymatic activity of type I DHNA-CoA synthases. The unique location of the bicarbonate ion in relation to the expected position of the substrate α-proton in the enzyme's active site suggests a critical catalytic role for the anionic cofactor as a catalytic base in enolate formation.     

VKORC1L1, an Enzyme Rescuing the Vitamin K 2,3-Epoxide Reductase Activity in Some Extrahepatic Tissues during Anticoagulation Therapy

Vitamin K is involved in the γ-carboxylation of the vitamin K-dependent proteins, and vitamin K epoxide is a by-product of this reaction. Due to the limited intake of vitamin K, its regeneration is necessary and involves vitamin K 2,3-epoxide reductase (VKOR) activity. This activity is known to be supported by VKORC1 protein, but recently a second gene, VKORC1L1, appears to be able to support this activity when the encoded protein is expressed in HEK293T cells. Nevertheless, this protein was described as being responsible for driving the vitamin K-mediated antioxidation pathways. In this paper we precisely analyzed the catalytic properties of VKORC1L1 when expressed in Pichia pastoris and more particularly its susceptibility to vitamin K antagonists. Vitamin K antagonists are also inhibitors of VKORC1L1, but this enzyme appears to be 50-fold more resistant to vitamin K antagonists than VKORC1. The expression of Vkorc1l1 mRNA was observed in all tissues assayed, i.e. in C57BL/6 wild type and VKORC1-deficient mouse liver, lung, and testis and rat liver, lung, brain, kidney, testis, and osteoblastic cells. The characterization of VKOR activity in extrahepatic tissues demonstrated that a part of the VKOR activity, more or less important according to the tissue, may be supported by VKORC1L1 enzyme especially in testis, lung, and osteoblasts. Therefore, the involvement of VKORC1L1 in VKOR activity partly explains the low susceptibility of some extrahepatic tissues to vitamin K antagonists and the lack of effects of vitamin K antagonists on the functionality of the vitamin K-dependent protein produced by extrahepatic tissues such as matrix Gla protein or osteocalcin.     

Immunopurification and Immunocharacterization of the Glucosinolate Biosynthetic Enzyme Thiohydroximate S-Glucosyltransferase

Preparing homogeneous UDP-glucose:thiohydroximate S-glucosyltransferase (S-GT), the penultimate biosynthetic enzyme of glucosinolates, by standard chromatographic methods has yielded too little protein for adequate purity evaluation, identity verification, and structural analysis. The low yields were apparently due to low abundance in source tissues, aggravated by enzyme instability. Here we describe an immunological method for purification of workable quantities from florets of Brassica oleracea ssp. botrytis (cauliflower). Florets that had undergone browning due to exposure to sunlight contained higher S-GT activities than are normally found in Brassica tissues. S-GT was adsorbed from crude tissue extracts onto an agarose-monoclonal antibody complex. Elution from the complex required harsh alkaline conditions (pH 11.5), giving extremely variable activity recoveries (maximum 20%). The eluate contained two proteins that could be separated readily by preparative polyacrylamide gel electrophoresis or anion-exchange chromatography. The overall S-GT protein recovery was estimated at less than 200 [mu]g/kg of cauliflower tissue. Molecular weight determinations with homogeneous cauliflower S-GT gave relative molecular weight (Mr) values of 55,500 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 57,600 by gel chromatography; isoenzymes with isoelectric point values of 4.80 and 4.95 were identified. A polyclonal antibody raised against denatured enzyme showed broad cross-reactivity in immunoblots with S-GT from a number of Brassica species and other crucifers. The monoclonal antibody that was used in the immunopurification was much more specific; it exclusively precipitated S-GT isoenzymes that had their genomic origin in the primary diploids B. oleracea and Brassica campestris. Thus, all of the S-GT was precipitated from the amphidiploid Brassica napus, which is a hybrid of B. orleracea and B. campestris. About half of the S-GT was precipitated from the amphidiploids Brassica carinata and Brassica juncea, which have B. oleracea and B. campestris as one of their parents, respectively. It was shown that the S-GT isoenzymes of B. juncea with Mr 55,500 and about 57,000 originate from the parents B. campestris and B. nigra, respectively.     

Menaquinone (Vitamin K2) Biosynthesis: Localization and Characterization of the menA Gene from Escherichia coli

A key reaction in the biosynthesis of menaquinone involves the conversion of the soluble bicyclic naphthalenoid compound 1,4-dihydroxy-2-naphthoic acid (DHNA) to the membrane-bound demethylmenaquinone. The enzyme catalyzing this reaction, DHNA-octaprenyltransferase, attaches a 40-carbon side chain to DHNA. The menA gene encoding this enzyme has been cloned and localized to a 2.0-kb region of the Escherichia coli genome between cytR and glpK. DNA sequence analysis of the cloned insert revealed a 308-codon open reading frame (ORF), which by deletion analyses was shown to restore anaerobic growth of a menA mutant. Reverse-phase high-performance liquid chromatography analysis of quinones extracted from the orf-complemented cells independently confirmed the restoration of menaquinone biosynthesis, and similarly, analyses of isolated cell membranes for DHNA octaprenyltransferase activity confirmed the introduction of the menA product into the orf-complemented menA mutant. The validity of an ORF-associated putative promoter sequence was confirmed by primer extension analyses.     

小鼠肝脏RNA编辑酶ADAR1 4种剪切体：克隆、表达及功能分析

RNA编辑是DNA转录为RNA后遗传信息发生改变的一种方式.A-to-IRNA编辑酶ADAR1(adenosinedeaminasethatactsonRNA1)具有将pre-mRNA中特定的腺嘌呤核苷转变为次黄嘌呤核苷的功能.通过RT-PCR技术从小鼠肝脏组织中克隆了小鼠A-to-IRNA编辑酶ADAR1的4种剪切体,采用荧光示踪技术研究其在细胞内定位,利用Bac-to-Bac杆状病毒表达系统构建了ADAR1重组杆状病毒并在sf9昆虫细胞内将其进行了表达,最后对表达产物进行了活性鉴定.结果发现,小鼠ADAR1在小鼠肝脏组织中主要以4种剪切方式存在,分别命名为ADAR1-La\Lb和ADAR1-Sa\Sb.这4种ADAR1剪切体在细胞内分布有着明显的区别,ADAR1-La\Lb主要分布于胞浆,而ADAR1-Sa\Sb主要分布于细胞核及核仁.Bac-to-Bac杆状病毒表达系统表达的4种ADAR1剪切体蛋白的双链RNA编辑活性明显不同,提示各个ADAR1剪切体的底物识别和特异性RNA编辑功能可能有所不同.ADAR1剪切体的克隆和表达以及它们在细胞内定位和编辑活性的差异的发现为进一步研究其结构和功能的关系及寻找它们的新底物奠定了基础.     

Menadione (Vitamin K3) Is a Catabolic Product of Oral Phylloquinone (Vitamin K1) in the Intestine and a Circulating Precursor of Tissue Menaquinone-4 (Vitamin K2) in Rats

Mice have the ability to convert dietary phylloquinone (vitamin K₁) into menaquinone-4 (vitamin K₂) and store the latter in tissues. A prenyltransferase enzyme, UbiA prenyltransferase domain-containing 1 (UBIAD1), is involved in this conversion. There is evidence that UBIAD1 has a weak side chain cleavage activity for phylloquinone but a strong prenylation activity for menadione (vitamin K₃), which has long been postulated as an intermediate in this conversion. Further evidence indicates that when intravenously administered in mice phylloquinone can enter into tissues but is not converted further to menaquinone-4. These findings raise the question whether phylloquinone is absorbed and delivered to tissues in its original form and converted to menaquinone-4 or whether it is converted to menadione in the intestine followed by delivery of menadione to tissues and subsequent conversion to menaquinone-4. To answer this question, we conducted cannulation experiments using stable isotope tracer technology in rats. We confirmed that the second pathway is correct on the basis of structural assignments and measurements of phylloquinone-derived menadione using high resolution MS analysis and a bioassay using recombinant UBIAD1 protein. Furthermore, high resolution MS and ¹H NMR analyses of the product generated from the incubation of menadione with recombinant UBIAD1 revealed that the hydroquinone, but not the quinone form of menadione, was an intermediate of the conversion. Taken together, these results provide unequivocal evidence that menadione is a catabolic product of oral phylloquinone and a major source of tissue menaquinone-4.     

Structure of a Membrane-Embedded Prenyltransferase Homologous to UBIAD1

Membrane-embedded prenyltransferases from the UbiA family catalyze the Mg²⁺-dependent transfer of a hydrophobic polyprenyl chain onto a variety of acceptor molecules and are involved in the synthesis of molecules that mediate electron transport, including Vitamin K and Coenzyme Q. In humans, missense mutations to the protein UbiA prenyltransferase domain-containing 1 (UBIAD1) are responsible for Schnyder crystalline corneal dystrophy, which is a genetic disease that causes blindness. Mechanistic understanding of this family of enzymes has been hampered by a lack of three-dimensional structures. We have solved structures of a UBIAD1 homolog from Archaeoglobus fulgidus, AfUbiA, in an unliganded form and bound to Mg²⁺ and two different isoprenyl diphosphates. Functional assays on MenA, a UbiA family member from E. coli, verified the importance of residues involved in Mg²⁺ and substrate binding. The structural and functional studies led us to propose a mechanism for the prenyl transfer reaction. Disease-causing mutations in UBIAD1 are clustered around the active site in AfUbiA, suggesting the mechanism of catalysis is conserved between the two homologs.     

建立维生素K_1微生物限度测定方法

目的:根据计数培养基适用性检查和计数方法适用性试验结果,建立维生素K1微生物限度测定方法。方法:以聚山梨酯80作为乳化剂,使维生素K1在p H 7.0无菌氯化钠-蛋白胨缓冲液中充分乳化。取1:20供试液1 m L,按平皿法分别用营养琼脂培养基和玫瑰红钠琼脂培养基培养,以计数细菌、霉菌和酵母菌。结果:验证所用培养基的菌落平均数均大于对照培养基上的70%,且菌落形态大小一致。稀释液对照组和试验组培养基上菌落平均数的回收率均大于70%,且菌落形态大小一致。三批维生素K1及其加速和长期稳定性样品中均未见菌落。结论:所选培养基适宜大肠埃希菌、金黄色葡萄球菌和白色念珠菌生长。且含聚山梨酯80的p H 7.0无菌氯化钠-蛋白胨缓冲液和维生素K1对所含大肠埃希菌、金黄色葡萄球菌和白色念珠菌无抑菌性。