Towards engineering increased pantothenate (vitamin B5) levels in plants

Pantothenate (vitamin B(5)) is the precursor of the 4'-phosphopantetheine moiety of coenzyme A and acyl-carrier protein. It is made by plants and microorganisms de novo, but is a dietary requirement for animals. The pantothenate biosynthetic pathway is well-established in bacteria, comprising four enzymic reactions catalysed by ketopantoate hydroxymethyltransferase (KPHMT), L: -aspartate-alpha-decarboxylase (ADC), pantothenate synthetase (PS) and ketopantoate reductase (KPR) encoded by panB, panD, panC and panE genes, respectively. In higher plants, the genes encoding the first (KPHMT) and last (PS) enzymes have been identified and characterised in several plant species. Commercially, pantothenate is chemically synthesised and used in vitamin supplements, feed additives and cosmetics. Biotransformation is an attractive alternative production system that would circumvent the expensive procedures of separating racemic intermediates. We explored the possibility of manipulating pantothenate biosynthesis in plants. Transgenic oilseed rape (Brassica napus) lines were generated in which the E. coli KPHMT and PS genes were expressed under a strong constitutive CaMV35SS promoter. No significant change of pantothenate levels in PS transgenic lines was observed. In contrast plants expressing KPHMT had elevated pantothenate levels in leaves, flowers siliques and seed in the range of 1.5-2.5 fold increase compared to the wild type plant. Seeds contained the highest vitamin content, indicating that they might be the ideal target for production purposes.     

Comparative analysis of the Escherichia coli ketopantoate hydroxymethyltransferase crystal structure confirms that it is a member of the (betaalpha)8 phosphoenolpyruvate/pyruvate superfamily

Escherichia coli ketopantoate hydroxymethyltransferase (KPHMT) catalyzes the first step in the biosynthesis pathway of pantothenate (vitamin B(5)), the transfer of a hydroxymethyl group onto alpha-ketoisovalerate. Here we describe a detailed comparative analysis of the KPHMT crystal structure and the identification of structural homologues, some of which have remarkable similarities in their active sites, modes of binding to substrates, and mechanisms. We show that KPHMT forms a family within the phosphoenolpyruvate/pyruvate superfamily. Based on the analysis, we propose that in this superfamily there should be a subdivision into two groups. This paper completes our structural analysis of the E. coli enzymes in the pantothenate pathway.     

Structure of E. coli ketopantoate hydroxymethyl transferase complexed with ketopantoate and Mg2+, solved by locating 160 selenomethionine sites

We report the crystal structure of E. coli ketopantoate hydroxymethyltransferase (KPHMT) at 1.9 A resolution, in complex with its product, ketopantoate. KPHMT catalyzes the first step in the biosynthesis of pantothenate (vitamin B(5)), the precursor of coenzyme A and the acyl carrier protein cofactor. The structure of the decameric enzyme was solved by multiwavelength anomalous dispersion to locate 160 selenomethionine sites and phase 560 kDa of protein, making it the largest structure solved by this approach. KPHMT adopts the (betaalpha)(8) barrel fold and is a member of the phosphoenolpyruvate/pyruvate superfamily. The active site contains a ketopantoate bidentately coordinated to Mg(2+). Similar binding is likely for the substrate, alpha-ketoisovalerate, orienting the C3 for deprotonation.     

The crystal structure of the first enzyme in the pantothenate biosynthetic pathway,ketopantoate hydroxymethyltransferase,from M tuberculosis

Ketopantoate hydroxymethyltransferase (KPHMT) catalyzes the first committed step in the biosynthesis of pantothenate, which is a precursor to coenzyme A and is required for penicillin biosynthesis. The crystal structure of KPHMT from Mycobacterium tuberculosis was determined by the single anomalous substitution (SAS) method at 2.8 A resolution. KPHMT adopts a structure that is a variation on the (beta/alpha) barrel fold, with a metal binding site proximal to the presumed catalytic site. The protein forms a decameric complex, with subunits in opposing pentameric rings held together by a swapping of their C-terminal alpha helices. The structure reveals KPHMT's membership in a small, recently discovered group of (beta/alpha) barrel enzymes that employ domain swapping to form a variety of oligomeric assemblies. The apparent conservation of certain detailed structural characteristics suggests that KPHMT is distantly related by divergent evolution to enzymes in unrelated pathways, including isocitrate lyase and phosphoenolpyruvate mutase.     

Molecular adaptation and allostery in plant pantothenate synthetases

Pantothenate synthetase catalyzes the ATP-dependent condensation of pantoate and beta-alanine to yield pantothenate, the essential precursor to coenzyme A. Bacterial and plant pantothenate synthetases are dimeric enzymes that share significant sequence identity. Here we show that the two-step reaction mechanism of pantothenate synthetase is conserved between the enzymes from Arabidopsis thaliana and Escherichia coli. Strikingly, though, the Arabidopsis enzyme exhibits large allosteric effects, whereas the Escherichia coli enzyme displays essentially non-allosteric behavior. Our data suggest that specific subunit contacts were selected and maintained in the plant lineage of the pantothenate synthetase protein family and that the resulting allosteric interactions are balanced for efficient catalysis at low pantoate levels. This is supported by mutations in the putative subunit interface of Arabidopsis pantothenate synthetase, which strongly attenuated or otherwise modified its allosteric properties but did not affect the dimeric state of the enzyme. At the molecular level, plant pantothenate synthetases exemplify functional adaptation through allostery and without alterations to the active site architecture. We propose that the allosteric behavior confers a selective advantage in the context of the subcellular compartmentation of pantothenate biosynthesis in plants.     

Crystal structure of uncleaved L-aspartate-alpha-decarboxylase from Mycobacterium tuberculosis

L-aspartate-alpha-decarboxylase (ADC) is a critical regulatory enzyme in the pantothenate biosynthetic pathway and belongs to a small class of self-cleaving and pyruvoyl-dependent amino acid decarboxylases. The expression level of ADC in Mycobacterium tuberculosis (Mtb) was confirmed by cDNA analysis, immunoblotting with an anti-ADC polyclonal antibody using whole cell lysate and immunoelectron microscopy. The recombinant ADC proenzyme from Mycobacterium tuberculosis (MtbADC) was overexpressed in E. coli and the protein structure was determined at 2.99 A resolution. The proteins fold into the double-psi beta-barrel structure. The subunits of the two tetramers (there are eight ADC molecules in the asymmetric unit) form pseudo fourfold rotational symmetry, similar to the E. coli ADC proenzyme structure. As pantothenate is synthesized in microorganisms, plants, and fungi but not in animals, structure elucidation of Mtb ADC is of substantial interest for structure-based drug development.     

Identification and characterization of an archaeal ketopantoate reductase and its involvement in regulation of coenzyme A biosynthesis

Coenzyme A (CoA) biosynthesis in bacteria and eukaryotes is regulated primarily by feedback inhibition towards pantothenate kinase (PanK). As most archaea utilize a modified route for CoA biosynthesis and do not harbour PanK, the mechanisms governing regulation of CoA biosynthesis are unknown. Here we performed genetic and biochemical studies on the ketopantoate reductase (KPR) from the hyperthermophilic archaeon Thermococcus kodakarensis. KPR catalyses the second step in CoA biosynthesis, the reduction of 2‐oxopantoate to pantoate. Gene disruption of TK1968, whose product was 20–29% identical to previously characterized KPRs from bacteria/eukaryotes, resulted in a strain with growth defects that were complemented by addition of pantoate. The TK1968 protein (Tk‐KPR) displayed reductase activity specific for 2‐oxopantoate and preferred NADH as the electron donor, distinct to the bacterial/eukaryotic NADPH‐dependent enzymes. Tk‐KPR activity decreased dramatically in the presence of CoA and KPR activity in cell‐free extracts was also inhibited by CoA. Kinetic studies indicated that CoA inhibits KPR by competing with NADH. Inhibition of ketopantoate hydroxymethyltransferase, the first enzyme of the pathway, by CoA was not observed. Our results suggest that CoA biosynthesis in T. kodakarensis is regulated by feedback inhibition of KPR, providing a feasible regulation mechanism of CoA biosynthesis in archaea.     

Plant cytosolic ribosomal protein S11 and chloroplast ribosomal protein CS17. Their primary structures and evolutionary relationships

We have isolated cDNA clones specific for Arabidopsis thaliana cytosolic ribosomal protein S11 and plastid ribosomal protein CS17, both of which are encoded in the nuclear genome, through the use of the corresponding soybean and pea cDNAs as probes, respectively. The nucleotide sequences of all four cDNAs were determined. The amino acid sequences derived from these cDNA sequences show that the soybean and A. thaliana S11 cDNAs encode proteins that are homologous to rat ribosomal protein S11 and that the pea and A. thaliana CS17 cDNAs encode proteins that are homologous to Escherichia coli ribosomal protein S17. The plant S11 cytosolic ribosomal proteins also show significant sequence similarity to both E. coli ribosomal protein S17 and plastid CS17 indicating that these are all related proteins. Comparison of A. thaliana CS17 with A. thaliana S11 and with E. coli S17 suggests that CS17 is more related to S17 than it is to S11. These results support the idea that the gene encoding CS17 was derived from a prokaryotic endosymbiont and not from a duplication of the eukaryotic S11 gene.     

Crystal structure of Escherichia coli ketopantoate reductase in a ternary complex with NADP+ and pantoate bound: substrate recognition, conformational change, and cooperativity

Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent reduction of ketopantoate to pantoate, an essential step for the biosynthesis of pantothenate (vitamin B5). Inhibitors of the enzymes of this pathway have been proposed as potential antibiotics or herbicides. Here we present the crystal structure of Escherichia coli KPR in a precatalytic ternary complex with NADP+ and pantoate bound, solved to 2.3 A of resolution. The asymmetric unit contains two protein molecules, each in a ternary complex; however, one is in a more closed conformation than the other. A hinge bending between the N- and C-terminal domains is observed, which triggers the switch of the essential Lys176 to form a key hydrogen bond with the C2 hydroxyl of pantoate. Pantoate forms additional interactions with conserved residues Ser244, Asn98, and Asn180 and with two conservatively varied residues, Asn194 and Asn241. The steady-state kinetics of active site mutants R31A, K72A, N98A, K176A, S244A, and E256A implicate Asn98 as well as Lys176 and Glu256 in the catalytic mechanism. Isothermal titration calorimetry studies with these mutants further demonstrate the importance of Ser244 for substrate binding and of Arg31 and Lys72 for cofactor binding. Further calorimetric studies show that KPR discriminates binding of ketopantoate against pantoate only with NADPH bound. This work provides insights into the roles of active site residues and conformational changes in substrate recognition and catalysis, leading to the proposal of a detailed molecular mechanism for KPR activity.     

The plant biotin synthase reaction. Identification and characterization of essential mitochondrial accessory protein components

In plants, the last step of the biotin biosynthetic pathway is localized in mitochondria. This chemically complex reaction is catalyzed by the biotin synthase protein, encoded by the bio2 gene in Arabidopsis thaliana. Unidentified mitochondrial proteins in addition to the bio2 gene product are obligatory for the reaction to occur. In order to identify these additional proteins, potato mitochondrial matrix was fractionated onto different successive chromatographic columns. Combination experiments using purified Bio2 protein and the resulting mitochondrial matrix subfractions together with a genomic based research allowed us to identify mitochondrial adrenodoxin, adrenodoxin reductase, and cysteine desulfurase (Nfs1) proteins as essential components for the plant biotin synthase reaction. Arabidopsis cDNAs encoding these proteins were cloned, and the corresponding proteins were expressed in Escherichia coli cells and purified. Purified recombinant adrenodoxin and adrenodoxin reductase proteins formed in vitro an efficient low potential electron transfer chain that interacted with the bio2 gene product to reconstitute a functional plant biotin synthase complex. Bio2 from Arabidopsis is the first identified protein partner for this specific plant mitochondrial redox chain.     

Molybdenum co-factor biosynthesis: the Arabidopsis thaliana cDNA cnx1 encodes a multifunctional two-domain protein homologous to a mammalian neuroprotein, the insect protein Cinnamon and three Escherichia coli proteins

The molybdenum co-factor (Moco) is an essential part of all eukaryotic molybdoenzymes. It is a molybdopterin and reveals the same principal structure in eubacteria, archaebacteria and eukaryotes. This paper reports the isolation of cnx1 , a cDNA clone of Arabidopsis thaliana which complements the Escherichia coli Moco mutant mogA . The mapping data of this cDNA correlate well with the mapping position of the A. thaliana molybdenum cofactor locus chl6 . As mutants in chl6 are known to be repairable by high concentrations of molybdate, the defective gene is very likely to be involved in the last step of Moco biosynthesis, that is, the insertion of molybdenum into molybdopterin. The protein encoded by cnx1 shows a two-domain structure: the N-terminal domain is homologous to the E. coli Moco protein MoeA, the C-terminal domain is homologous to the E. coli Moco proteins MoaB and MogA, respectively. These homologies show that part of the prokaryotic Moco biosynthetic pathway accomplished by monofunctional proteins in E. coli , is performed by a single multifunctional protein in eukaryotes. In addition Cnx1 is homologous to the eukaryotic proteins Gephyrin, a rat neuroprotein, and Cinnamon, a Drosophila protein with a function in Moco biosynthesis. These proteins also show a two-domain structure but the order of the domains is inversed as compared with Cnx1. Southern analysis indicates the existence of at least one further member, in addition to the cnx1 gene, of this novel gene family in the Arabidopsis genome.     

Cloning and expression of two novel aldo-keto reductases from Digitalis purpurea leaves.

The aldo-keto reductase (AKR) superfamily comprises proteins that catalyse mainly the reduction of carbonyl groups or carbon-carbon double bonds of a wide variety of substrates including steroids. Such types of reactions have been proposed to occur in the biosynthetic pathway of the cardiac glycosides produced by Digitalis plants. Two cDNAs encoding leaf-specific AKR proteins (DpAR1 and DpAR2) were isolated from a D. purpurea cDNA library using the rat Delta4-3-ketosteroid 5beta-reductase clone. Both cDNAs encode 315 amino acid proteins showing 98.4% identity. DpAR proteins present high identities (68-80%) with four Arabidopsis clones and a 67% identity with the aldose/aldehyde reductase from Medicago sativa. A molecular phylogenetic tree suggests that these seven proteins belong to a new subfamily of the AKR superfamily. Southern analysis indicated that DpARs are encoded by a family of at most five genes. RNA-blot analyses demonstrated that the expression of DpAR genes is developmentally regulated and is restricted to leaves. The expression of DpAR genes has also been induced by wounding, elevated salt concentrations, drought stress and heat-shock treatment. The isolated cDNAs were expressed in Escherichia coli and the recombinant proteins purified. The expressed enzymes present reductase activity not only for various sugars but also for steroids, preferring NADH as a cofactor. These studies indicate the presence of plant AKR proteins with ketosteroid reductase activity. The function of the enzymes in cardenolide biosynthesis is discussed.     

A plant‐specific in vitro ubiquitination analysis system

Protein ubiquitination requires the concerted action of three enzymes: ubiquitin‐activating enzyme (E1), ubiquitin‐conjugating enzyme (E2) and ubiquitin ligase (E3). These ubiquitination enzymes belong to an abundant protein family that is encoded in all eukaryotic genomes. Describing their biochemical characteristics is an important part of their functional analysis. It has been recognized that various E2/E3 specificities exist, and that detection of E3 ubiquitination activity in vitro may depend on the recruitment of E2s. Here, we describe the development of an in vitro ubiquitination system based on proteins encoded by genes from Arabidopsis. It includes most varieties of Arabidopsis E2 proteins, which are tested with several RING‐finger type E3 ligases. This system permits determination of E3 activity in combination with most of the E2 sub‐groups that have been identified in the Arabidopsis genome. At the same time, E2/E3 specificities have also been explored. The components used in this system are all from plants, particularly Arabidopsis, making it very suitable for ubiquitination assays of plant proteins. Some E2 proteins that are not easily expressed in Escherichia coli were transiently expressed and purified from plants before use in ubiquitination assays. This system is also adaptable to proteins of species other than plants. In this system, we also analyzed two mutated forms of ubiquitin, K48R and K63R, to detect various types of ubiquitin conjugation.     

Coenzyme A biosynthesis: reconstruction of the pathway in archaea and an evolutionary scenario based on comparative genomics

Coenzyme A (CoA) holds a central position in cellular metabolism and therefore can be assumed to be an ancient molecule. Starting from the known E. coli and human enzymes required for the biosynthesis of CoA, phylogenetic profiles and chromosomal proximity methods enabled an almost complete reconstruction of archaeal CoA biosynthesis. This includes the identification of strong candidates for archaeal pantothenate synthetase and pantothenate kinase, which are unrelated to the corresponding bacterial or eukaryotic enzymes. According to this reconstruction, the topology of CoA synthesis from common precursors is essentially conserved across the three domains of life. The CoA pathway is conserved to varying degrees in eukaryotic pathogens like Giardia lamblia or Plasmodium falciparum, indicating that these pathogens have individual uptake-mechanisms for different CoA precursors. Phylogenetic analysis and phyletic distribution of the CoA biosynthetic enzymes suggest that the enzymes required for the synthesis of phosphopantothenate were recruited independently in the bacterial and archaeal lineages by convergent evolution, and that eukaryotes inherited the genes for the synthesis of pantothenate (vitamin B5) from bacteria. Homologues to bacterial enzymes involved in pantothenate biosynthesis are present in a subset of archaeal genomes. The phylogenies of these enzymes indicate that they were acquired from bacterial thermophiles through horizontal gene transfer. Monophyly can be inferred for each of the enzymes catalyzing the four ultimate steps of CoA synthesis, the conversion of phosphopantothenate into CoA. The results support the notion that CoA was initially synthesized from a prebiotic precursor, most likely pantothenate or a related compound.