Folate Metabolism Gene 5,10-Methylenetetrahydrofolate Reductase (MTHFR) Is Associated with ADHD in Myelomeningocele Patients

The objective of this study was to examine the relation between the 5, 10-methylenetetrahydrofolate reductase (MTHFR) gene and behaviors related to attention- deficit/hyperactivity disorder (ADHD) in individuals with myelomeningocele. The rationale for the study was twofold: folate metabolizing genes, (e.g. MTHFR), are important not only in the etiology of neural tube defects but are also critical to cognitive function; and individuals with myelomeningocele have an elevated incidence of ADHD. Here, we tested 478 individuals with myelomeningocele for attention-deficit hyperactivity disorder behavior using the Swanson Nolan Achenbach Pelham-IV ADHD rating scale. Myelomeningocele participants in this group for whom DNAs were available were genotyped for seven single nucleotide polymorphisms (SNPs) in the MTHFR gene. The SNPs were evaluated for an association with manifestation of the ADHD phenotype in children with myelomeningocele. The data show that 28.7% of myelomeningocele participants exhibit rating scale elevations consistent with ADHD; of these 70.1% had scores consistent with the predominantly inattentive subtype. In addition, we also show a positive association between the SNP rs4846049 in the 3′-untranslated region of the MTHFR gene and the attention-deficit hyperactivity disorder phenotype in myelomeningocele participants. These results lend further support to the finding that behavior related to ADHD is more prevalent in patients with myelomeningocele than in the general population. These data also indicate the potential importance of the MTHFR gene in the etiology of the ADHD phenotype.     

In Vitro Studies of 5,10-Methylenetetrahydrofolate Reductase: Inhibition by Folate Derivatives, Folate Antagonists, and Monoamine Derivatives

Abstract: Folate monoglutamates and folate antagonists have an inhibitory action on the activity of 5, 10-methylenetetrahydrofolate reductase in rat brain. The type of inhibition was studied for dihydrofolic acid using the Lineweaver–Burk transformation. Some of the monoamine alkaloids, the in vitro products of 5, 10-methylenetetrahydrofolate reductase, have either a stimulatory or inhibitory effect on the enzyme activity.     

负调节基因nsdA在链霉菌中同源性及激活沉默抗生素合成基因簇的研究

nsdA基因是在天蓝色链霉菌中发现的抗生素合成负调控基因。以nsdA基因片段为探针,通过Southern杂交发现nsdA存在于多种链霉菌中。根据天蓝色链霉菌和阿维链霉菌的nsdA序列设计PCR引物,扩增多种链霉菌中nsdA基因并测序。发现在不同链霉菌中nsdA基因的相似性高达77%~100%。其中变铅青链霉菌与天蓝色链霉菌A3(2)的nsdA序列100%一致。变铅青链霉菌通常不合成放线紫红素,中断nsdA获得的突变菌株WQ2能够合成放线紫红素;在WQ2中重新引入野生型nsdA,又失去产抗生素能力。表明nsdA的中断可以激活变铅青链霉菌中沉默的放线紫红素生物合成基因簇的表达;nsdA的广泛存在及其序列高度保守则提示可以尝试用于这些菌种的抗生素高产育种。     

Gene Targeting in Penicillium chrysogenum: Disruption of the lys2 Gene Leads to Penicillin Overproduction

Two strategies have been used for targeted integration at the lys2 locus of Penicillium chrysogenum. In the first strategy the disruption of lys2 was obtained by a single crossing over between the endogenous lys2 and a fragment of the same gene located in an integrative plasmid. lys2-disrupted mutants were obtained with 1.6% efficiency when the lys2 homologous region was 4.9 kb, but no homologous integration was observed with constructions containing a shorter homologous region. Similarly, lys2-disrupted mutants were obtained by a double crossing over (gene replacement) with an efficiency of 0.14% by using two lys2 homologous regions of 4.3 and 3.0 kb flanking the pyrG marker. No homologous recombination was observed when the selectable marker was flanked by short lys2 homologous DNA fragments. The disruption of lys2 was confirmed by Southern blot analysis of three different lysine auxotrophs obtained by a single crossing over or gene replacement. The lys2-disrupted mutants lacked α-aminoadipate reductase activity (encoded by lys2) and showed specific penicillin yields double those of the parental nondisrupted strain, Wis 54-1255. The α-aminoadipic acid precursor is channelled to penicillin biosynthesis by blocking the lysine biosynthesis branch at the α-aminoadipate reductase level.     

Methionine or Folate and Phosphoenolpyruvate in the Biosynthesis of Threonine in Crithidia fasciculata1

SYNOPSIS. Crithidia fasciculata can synthesize threonine but it lacks an aspartokinase. The carbons of threonine may be derived from methionine when it is present in the medium. However, methionine can be synthesized by the organism provided organic sulfur is present. When both methionine and threonine are omitted from the medium, growth will occur if cysteine and high levels of folate are present. The compound common to both methionine and threonine under these conditions, α-keto-γ-hydroxy-butyrate, is derived from phosphoenolpyruvate (PEP) and the β-carbon of serine. High levels of folate are required for this coupling reaction, which is carried out by what may be called phosphoenolpyruvate-tetrahydrofolate hydroxymethyltransferase. By the use of radioactive tracers, both in growth experiments and in cell-free preparations, virtually all of the intermediates in these two series of reactions were identified.     

Disruption of the Selenocysteine Lyase-Mediated Selenium Recycling Pathway Leads to Metabolic Syndrome in Mice

Selenium (Se) is an essential trace element used for biosynthesis of selenoproteins and is acquired either through diet or cellular recycling mechanisms. Selenocysteine lyase (Scly) is the enzyme that supplies Se for selenoprotein biosynthesis via decomposition of the amino acid selenocysteine (Sec). Knockout (KO) of Scly in a mouse affected hepatic glucose and lipid homeostasis. Mice lacking Scly and raised on an Se-adequate diet exhibit hyperinsulinemia, hyperleptinemia, glucose intolerance, and hepatic steatosis, with increased hepatic oxidative stress, but maintain selenoprotein levels and circulating Se status. Insulin challenge of Scly KO mice results in attenuated Akt phosphorylation but does not decrease phosphorylation levels of AMP kinase alpha (AMPKα). Upon dietary Se restriction, Scly KO animals develop several characteristics of metabolic syndrome, such as obesity, fatty liver, and hypercholesterolemia, with aggravated hyperleptinemia, hyperinsulinemia, and glucose intolerance. Hepatic glutathione peroxidase 1 (GPx1) and selenoprotein S (SelS) production and circulating selenoprotein P (Sepp1) levels are significantly diminished. Scly disruption increases the levels of insulin-signaling inhibitor PTP1B. Our results suggest a dependence of glucose and lipid homeostasis on Scly activity. These findings connect Se and energy metabolism and demonstrate for the first time a unique physiological role of Scly in an animal model.     

Disruption of the ste22 Gene Encoding a Glycosyltransferase and Its Function in Biosynthesis of Ebosin in Streptomyces sp. 139

Streptomyces sp.139 produces an exopolysaccharide (EPS) designated Ebosin with remarkable anti-rheumatic arthritis activity in vivo. The ste (Streptomyces eps) gene cluster required for Ebosin biosynthesis has been identified. According to similarities with other proteins in the database, ste22 shows high homology with glycosyltransferases originated from different microorganisms. In this study, the ste22 gene was disrupted by double crossover via homologous recombination. The EPS produced by the mutant strain Streptomyces sp.139 (ste22⁻) has a different monosaccharide composition profile in comparison with that of Ebosin. This derivative of Ebosin retained the original antagonistic activity of IL-1R in vitro but lost the bioactivities of anti-inflammation and pain relief in vivo.     

Identification and Characterization of the Missing Pyrimidine Reductase in the Plant Riboflavin Biosynthesis Pathway

Riboflavin (vitamin B₂) is the precursor of the flavin coenzymes flavin mononucleotide and flavin adenine dinucleotide. In Escherichia coli and other bacteria, sequential deamination and reduction steps in riboflavin biosynthesis are catalyzed by RibD, a bifunctional protein with distinct pyrimidine deaminase and reductase domains. Plants have two diverged RibD homologs, PyrD and PyrR; PyrR proteins have an extra carboxyl-terminal domain (COG3236) of unknown function. Arabidopsis (Arabidopsis thaliana) PyrD (encoded by At4g20960) is known to be a monofunctional pyrimidine deaminase, but no pyrimidine reductase has been identified. Bioinformatic analyses indicated that plant PyrR proteins have a catalytically competent reductase domain but lack essential zinc-binding residues in the deaminase domain, and that the Arabidopsis PyrR gene (At3g47390) is coexpressed with riboflavin synthesis genes. These observations imply that PyrR is a pyrimidine reductase without deaminase activity. Consistent with this inference, Arabidopsis or maize (Zea mays) PyrR (At3g47390 or GRMZM2G090068) restored riboflavin prototrophy to an E. coli ribD deletant strain when coexpressed with the corresponding PyrD protein (At4g20960 or GRMZM2G320099) but not when expressed alone; the COG3236 domain was unnecessary for complementing activity. Furthermore, recombinant maize PyrR mediated NAD(P)H-dependent pyrimidine reduction in vitro. Import assays with pea (Pisum sativum) chloroplasts showed that PyrR and PyrD are taken up and proteolytically processed. Ablation of the maize PyrR gene caused early seed lethality. These data argue that PyrR is the missing plant pyrimidine reductase, that it is plastid localized, and that it is essential. The role of the COG3236 domain remains mysterious; no evidence was obtained for the possibility that it catalyzes the dephosphorylation that follows pyrimidine reduction.Riboflavin is the substrate for biosynthesis of the essential flavocoenzymes FMN and FAD, which occur in all kingdoms of life and have roles in diverse redox reactions as well as in other processes such as DNA repair, light sensing, and bioluminescence (Fischer and Bacher, 2005). Plants and many microorganisms can synthesize riboflavin, but humans and other animals cannot, so they must obtain it from the diet (Powers, 2003). Plant foods are important sources of riboflavin for humans, and the riboflavin pathway is a target for engineering biofortified crops (Fitzpatrick et al., 2012).Riboflavin biosynthesis proceeds via the same pathway in bacteria and plants (Fischer and Bacher, 2005; Roje, 2007). This pathway starts from GTP, which is converted by GTP cyclohydrolase II (named RibA in Escherichia coli) to the pyrimidine derivative 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-P. Deamination of the pyrimidine ring then yields 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5′-P, and subsequent reduction of the ribosyl moiety gives 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-P. After dephosphorylation, this product is condensed with 3,4-dihydroxy-2-butanone 4-P to give 6,7-dimethyl-8-ribityllumazine, whose dismutation yields riboflavin. Figure 1 shows the first four steps of this pathway.Open in a separate windowFigure 1.The first four steps of the riboflavin biosynthesis pathway in bacteria and plants. The enzymes involved are GTP cyclohydrolase II (RibA), pyrimidine deaminase (Deam), pyrimidine reductase (Red), and a specific phosphatase (Pase). Enzymes for which the plant genes are not known are colored red. Intermediates are as follows: 1, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-P; 2, 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5′-P; 3, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-P; 4, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione.In E. coli, the deamination and reduction steps are catalyzed by a single bifunctional enzyme, RibD, which has N-terminal deaminase and C-terminal reductase domains that retain their respective activities when expressed separately (Richter et al., 1997; Magalhães et al., 2008). The situation in plants seems superficially similar but is in fact more complex (Gerdes et al., 2012). The bidomain bacterial RibD protein has two types of homologs in plants (Fischer et al., 2004; Chatwell et al., 2006; Chen et al., 2006), here called PyrD and PyrR, both with apparent deaminase and reductase domains (Fig. 2A). Only PyrD, represented by At4g20960, has been studied biochemically; it was found to have pyrimidine deaminase but not reductase activity (Fischer et al., 2004). The function of PyrR, represented by At3g47390, is unknown, although it has been inferred to have reductase activity (Chatwell et al., 2006; Chen et al., 2006; Ouyang et al., 2010) and perhaps to lack deaminase activity (Ouyang et al., 2010). Another mystery surrounding PyrR proteins is the presence of an extra C-terminal domain of unknown function (COG3236 in the Clusters of Orthologous Groups database; Fig. 2A); this domain occurs as a stand-alone protein in many bacteria. One possibility is that it catalyzes the dephosphorylation that follows the pyrimidine reduction step in the pathway (Fig. 1). The phosphatase involved is most likely substrate specific, but it has not been identified in plants or any other organism (Roje, 2007; Gerdes et al., 2012), and genes for enzymes in the same pathway, especially for successive steps, are quite commonly fused (Suhre, 2007). A mutation (phs1) that deleted the COG3236 domain from Arabidopsis (Arabidopsis thaliana) PyrR resulted in a photosensitive phenotype that could be rescued by supplied FAD (Ouyang et al., 2010).Open in a separate windowFigure 2.Structure and phylogeny of plant PyrD and PyrR proteins. A, Domain architectures. The examples shown are Arabidopsis At4g20960 and At3g47390; the predicted plastid targeting peptide (TP) varies in length between species. B, Phylogenetic tree of PyrD and PyrR proteins. Sequences were aligned with ClustalW; the tree was built by the neighbor-joining method with MEGA5. Bootstrap values (%) for 1,000 replicates are next to the nodes. Only the tree topology is shown. Note that the PyrD proteins of green algae (underlined) lack a reductase domain. C, Alignments showing the conservation of the zinc-binding residues (arrowheads) in the deaminase domain of PyrD but not PyrR proteins and the conservation of the predicted substrate-binding residues (asterisks) in the reductase domain of PyrR but not PyrD proteins. The deaminase sequences correspond to residues 45 to 85 of B. subtilis RibD (synonym RibG); the reductase sequences correspond to residues 150 to 210 and (separated by dots) 288 to 292 of B. subtilis RibD. Identical zinc- or substrate-binding residues are black, and conservative replacements are gray. Dashes indicate gaps that maximize the alignment.The plant riboflavin synthesis pathway is considered to be plastidial (Roje, 2007), but this location is based almost solely on bioinformatics and high-throughput proteome analyses (Gerdes et al., 2012). In only one case is there more definitive experimental support: in vitro chloroplast import data for the pathway’s penultimate enzyme, 6,7-dimethyl-8-ribityllumazine synthase (Jordan et al., 1999). Similarly, clear genetic support for the function of most plant riboflavin synthesis enzymes is lacking (Gerdes et al., 2012), the exception being an Arabidopsis RibA homolog (Hedtke and Grimm, 2009).The work reported here established, using maize (Zea mays) and Arabidopsis, that PyrR is indeed the missing pyrimidine reductase, that it lacks deaminase activity, and that its COG3236 domain is not essential for pyrimidine reductase activity and most likely lacks phosphatase activity. We also demonstrated the import of PyrR and PyrD into chloroplasts in vitro and confirmed that the gene for PyrR is essential.     

Development of Antibiotic-Overproducing Strains by Site-Directed Mutagenesis of the rpsL Gene in Streptomyces lividans

Certain rpsL (which encodes the ribosomal protein S12) mutations that confer resistance to streptomycin markedly activate the production of antibiotics in Streptomyces spp. These rpsL mutations are known to be located in the two conserved regions within the S12 protein. To understand the roles of these two regions in the activation of silent genes, we used site-directed mutagenesis to generate eight novel mutations in addition to an already known (K88E) mutation that is capable of activating antibiotic production in Streptomyces lividans. Of these mutants, two (L90K and R94G) activated antibiotic production much more than the K88E mutant. Neither the L90K nor the R94G mutation conferred an increase in the level of resistance to streptomycin and paromomycin. Our results demonstrate the efficacy of the site-directed mutagenesis technique for strain improvement.     

普那霉素生物合成相关基因Spr1的功能

以与普那霉素生物合成密切相关的新基因Afsk-like为探针,从始旋链霉菌F618基因组文库中筛选得到含有约8 kb的DNA片段.经测序分析表明,其上含有1个具有1 146个核苷酸的完整可阅读框,该基因被命名为Spr1(HQ450023),推测其编码1个含381个氨基酸的蛋白质产物.经Blastp程序进行分析得知,该基...     

The Gene Cluster for Spectinomycin Biosynthesis and the Aminoglycoside-Resistance Function of spcM in Streptomyces spectabilis

The gene cluster for spectinomycin biosynthesis from Streptomyces spectabilis was analyzed completely and registered under the accession number EU255259 at the National Center for Biotechnology Information. Based on sequence analysis, spcM of the S. spectabilis cluster is the only methyltransferase candidate required for methylation in spectinomycin biosynthesis. It has high similarity with the conserved domain of DNA methylase, which contains both N-4 cytosine-specific DNA methylases and N-6 adenine-specific DNA methylases. Nucleotide methylation can provide antibiotic resistance, such as 16S rRNA methyltransferase, to Enterobacteriaceae. We therefore tested a hypothesis that SpcM offers aminoglycoside resistance to bacteria. The heterologous expression of spcM in Escherichia coli and S. lividans enhanced resistance against spectinomycin and its relative aminoglycoside antibiotics. We therefore propose that one of the functions of SpcM may be conferring aminoglycoside antibiotic resistance to cells.     

Cloning of the Thermomonospora fusca Endoglucanase E2 Gene in Streptomyces lividans: Affinity Purification and Functional Domains of the Cloned Gene Product

Thermomonospora fusca YX grown in the presence of cellulose produces a number of β-1-4-endoglucanases, some of which bind to microcrystalline cellulose. By using a multicopy plasmid, pIJ702, a gene coding for one of these enzymes (E2) was cloned into Streptomyces lividans and then mobilized into both Escherichia coli and Streptomyces albus. The gene was localized to a 1.6-kilobase PvuII-ClaI segment of the originally cloned 3.0-kilobase SstI fragment of Thermomonospora DNA. The culture supernatants of Streptomyces transformants contain a major endoglucanase that cross-reacts with antibody against Thermomonospora cellulase E2 and has the same molecular weight (43,000) as T. fusca E2. This protein binds quickly and tightly to Avicel, from which it can be eluted with guanidine hydrochloride but not with water. It also binds to filter paper but at a slower rate than to Avicel. Several large proteolytic degradation products of this enzyme generated in vivo lose the ability to bind to Avicel and have higher activity on carboxymethyl cellulose than the native enzyme. Other smaller products bind to Avicel but lack activity. A weak cellobiose-binding site not observed in the native enzyme was present in one of the degradation products. In E. coli, the cloned gene produced a cellulase that also binds tightly to Avicel but appeared to be slightly larger than T. fusca E2. The activity of intact E2 from all organisms can be inactivated by Hg²⁺ ions. Dithiothreitol protected against Hg²⁺ inactivation and reactivated both unbound and Avicel-bound Hg²⁺-inhibited E2, but at different rates.     

抗生素中L-玫红糖的生物合成基因簇结构与其合成途径

抗生素的生物合成过程中普遍存在被脱氧糖糖基化的现象。糖基的存在可以增加抗生素的溶解度和稳定性．提高抗生素的生物活性。L-玫红糖是6-脱氧己糖家族中的一种三脱氧葡萄糖。目前已有5种含有玫红糖及其衍生物的抗生素完成了糖基生物合成基因簇克隆及测序。研究结果表明L-玫红糖的生物合成基因簇有了一定的分化．但在基因结构上仍有较大的保守性。L-玫红糖的生物合成主要包括形成dNDP-葡萄糖、脱水、脱氧、酮基还原和异构等反应,其衍生物的合成还包括甲基化、酰基化等;这被人们普遍认可,但在一些细节上,如是否形成中间体dNDP-3,4-二酮-2,6-双脱氧-D-葡萄糖,以及糖基的异构何时发生等问题还存在分歧,仍需进一步研究。     

The Streptomyces peucetius dpsC Gene Determines the Choice of Starter Unit in Biosynthesis of the Daunorubicin Polyketide

The starter unit used in the biosynthesis of daunorubicin is propionyl coenzyme A (CoA) rather than acetyl-CoA, which is used in the production of most of the bacterial aromatic polyketides studied to date. In the daunorubicin biosynthesis gene cluster of Streptomyces peucetius, directly downstream of the genes encoding the beta-ketoacyl:acyl carrier protein synthase subunits, are two genes, dpsC and dpsD, encoding proteins that are believed to function as the starter unit-specifying enzymes. Recombinant strains containing plasmids carrying dpsC and dpsD, in addition to other daunorubicin polyketide synthase (PKS) genes, incorporate the correct starter unit into polyketides made by these genes, suggesting that, contrary to earlier reports, the enzymes encoded by dpsC and dpsD play a crucial role in starter unit specification. Additionally, the results of a cell-free synthesis of 21-carbon polyketides from propionyl-CoA and malonyl-CoA that used the protein extracts of recombinant strains carrying other daunorubicin PKS genes to which purified DpsC was added suggest that this enzyme has the primary role in starter unit discrimination for daunorubicin biosynthesis.     

New Rimocidin/CE-108 Derivatives Obtained by a Crotonyl-CoA Carboxylase/Reductase Gene Disruption in Streptomyces diastaticus var. 108: Substrates for the Polyene Carboxamide Synthase PcsA

The rimJ gene, which codes for a crotonyl-CoA carboxylase/reductase, lies within the biosynthetic gene cluster for two polyketides belonging to the polyene macrolide group (CE-108 and rimocidin) produced by Streptomyces diastaticus var. 108. Disruption of rimJ by insertional inactivation gave rise to a recombinant strain overproducing new polyene derivatives besides the parental CE-108 (2a) and rimocidin (4a). The structure elucidation of one of them, CE-108D (3a), confirmed the incorporation of an alternative extender unit for elongation step 13. Other compounds were also overproduced in the fermentation broth of rimJ disruptant. The new compounds are in vivo substrates for the previously described polyene carboxamide synthase PcsA. The rimJ disruptant strain, constitutively expressing the pcsA gene, allowed the overproduction of CE-108E (3b), the corresponding carboxamide derivative of CE-108D (3a), with improved pharmacological properties.     

Structure and Function of sanV: A Gene Involved in Nikkomycin Biosynthesis of Streptomyces ansochromogenes

A 6.3-kb BamHI-BglII DNA fragment was cloned from cos20 by using chromosome walking strategy. It was partially sequenced with the result that there is a possible ORF of 1272 nucleotides. The ORF designated sanV was deposited in GenBank under accession no. AF469955. Database search indicated that the deduced protein of sanV shows 28% identity and 44% similarity over 405 amino acid residues to the large component (E) of glutamate mutases from Clostridium cochlearium. Gene disruption was performed to study the function of sanV. It was found that sanV disruptants exhibited much poorer inhibition to the test strain than the wild-type S. ansochromogenes 7100. Furthermore, HPLC analysis indicated that the sanV disruptants almost did not produce nikkomycins X and Z, whereas they accumulated new nikkomycins O_x and O_z, which revealed that sanV is an important gene involved in the biosynthesis of the peptidyl moiety of nikkomycins. Received: 13 June 2002 / Accepted: 27 July 2002