Metabolic fate of L-lactaldehyde derived from an alternative L-rhamnose pathway

Fungal Pichia stipitis and bacterial Azotobacter vinelandii possess an alternative pathway of L-rhamnose metabolism, which is different from the known bacterial pathway. In a previous study (Watanabe S, Saimura M & Makino K (2008) Eukaryotic and bacterial gene clusters related to an alternative pathway of non-phosphorylated L-rhamnose metabolism. J Biol Chem283, 20372-20382), we identified and characterized the gene clusters encoding the four metabolic enzymes [L-rhamnose 1-dehydrogenase (LRA1), L-rhamnono-gamma-lactonase (LRA2), L-rhamnonate dehydratase (LRA3) and l-2-keto-3-deoxyrhamnonate aldolase (LRA4)]. In the known and alternative L-rhamnose pathways, L-lactaldehyde is commonly produced from l-2-keto-3-deoxyrhamnonate and L-rhamnulose 1-phosphate by each specific aldolase, respectively. To estimate the metabolic fate of L-lactaldehyde in fungi, we purified L-lactaldehyde dehydrogenase (LADH) from P. stipitis cells L-rhamnose-grown to homogeneity, and identified the gene encoding this enzyme (PsLADH) by matrix-assisted laser desorption ionization-quadruple ion trap-time of flight mass spectrometry. In contrast, LADH of A. vinelandii (AvLADH) was clustered with the LRA1-4 gene on the genome. Physiological characterization using recombinant enzymes revealed that, of the tested aldehyde substrates, L-lactaldehyde is the best substrate for both PsLADH and AvLADH, and that PsLADH shows broad substrate specificity and relaxed coenzyme specificity compared with AvLADH. In the phylogenetic tree of the aldehyde dehydrogenase superfamily, PsLADH is poorly related to the known bacterial LADHs, including that of Escherichia coli (EcLADH). However, despite its involvement in different L-rhamnose metabolism, AvLADH belongs to the same subfamily as EcLADH. This suggests that the substrate specificities for L-lactaldehyde between fungal and bacterial LADHs have been acquired independently.     

Metabolic engineering provides insight into the regulation of thiamin biosynthesis in plants

Thiamin (or thiamine) is a water-soluble B-vitamin (B1), which is required, in the form of thiamin pyrophosphate, as an essential cofactor in crucial carbon metabolism reactions in all forms of life. To ensure adequate metabolic functioning, humans rely on a sufficient dietary supply of thiamin. Increasing thiamin levels in plants via metabolic engineering is a powerful strategy to alleviate vitamin B1 malnutrition and thus improve global human health. These engineering strategies rely on comprehensive knowledge of plant thiamin metabolism and its regulation. Here, multiple metabolic engineering strategies were examined in the model plant Arabidopsis thaliana. This was achieved by constitutive overexpression of the three biosynthesis genes responsible for B1 synthesis, HMP-P synthase (THIC), HET-P synthase (THI1), and HMP-P kinase/TMP pyrophosphorylase (TH1), either separate or in combination. By monitoring the levels of thiamin, its phosphorylated entities, and its biosynthetic intermediates, we gained insight into the effect of either strategy on thiamin biosynthesis. Moreover, expression analysis of thiamin biosynthesis genes showed the plant’s intriguing ability to respond to alterations in the pathway. Overall, we revealed the necessity to balance the pyrimidine and thiazole branches of thiamin biosynthesis and assessed its biosynthetic intermediates. Furthermore, the accumulation of nonphosphorylated intermediates demonstrated the inefficiency of endogenous thiamin salvage mechanisms. These results serve as guidelines in the development of novel thiamin metabolic engineering strategies.     

Aerobic metabolism of phenylacetic acids in Azoarcus evansii

The aerobic metabolism of phenylacetic acid (PA) and 4-hydroxyphenylacetic acid (4-OHPA) was investigated in the beta-proteobacterium Azoarcus evansii. Evidence for the existence of two independent catabolic pathways for PA and 4-OHPA is presented. 4-OHPA metabolism involves the formation of 2,5-dihydroxyphenylacetate (homogentisate) and maleylacetoacetate catalyzed by specifically induced 4-OHPA 1-monooxygenase and homogentisate 1,2-dioxygenase. The metabolism of PA starts by its activation to phenylacetyl-CoA (PA-CoA) via an aerobically induced phenylacetate-coenzyme A ligase. Phenylalanine (Phe) aerobic metabolism in this bacterium proceeds also via PA and PA-CoA. Whole cells of A. evansii transformed [1-(14)C]PA to (14)C-phenylacetyl-CoA and subsequently to a number of unknown labeled products, which were also observed in PA-degrading bacteria from different phylogenetic groups, i.e. Escherichia coli, Rhodopseudomonas palustrisand Bacillus stearothermophilus. A chromosomal region from A. evansiiof 11.5 kb containing a cluster of 11 phenylacetic acid catabolic ( paa) genes ( paaYZGHIKABCDE) was sequenced and characterized. The derived gene products were similar to the characterized putative gene products involved in PA catabolism in E. coli and Pseudomonas putida and to other putative PA catabolic gene products of diverse bacteria. RT-PCR analysis of the paa genes of A. evansiigrowing aerobically with PA showed a probable organization of the paa genes in three operons. The similarity of the PA metabolic products pattern and of gene sequences suggests a common aerobic bacterial PA pathway.     

Molecular organization of the shikimate pathway in higher plants

The shikimate pathway produces the three proteinogenic aromatic amino acids, phenylalanine, tyrosine and tryptophan, which are, in addition to several intermediates of the shikimate pathway, intermediates in the biosynthesis of numerous aromatic natural products in higher plants. While there is only little difference in the sequence of the chemical reactions of the pathway in bacteria, fungi and plants, considerable differences exist in the organization and regulation of the shikimate pathway in plants, fungi and bacteria. The recent isolation and characterization of cDNAs and genes coding for enzymes of the shikimate pathway in higher plants have confirmed that plastids are the major, if not only site of aromatic amino acid biosynthesis in plants. Furthermore, the observed differential spatial and temporal expression of genes coding for isozymes of the pathway indicates a complex regulation that we are only beginning to understand.     

Identification of the Entner-Doudoroff pathway in an antibiotic-producing actinomycete species

The metabolic network of the central carbon metabolism represents the backbone of cellular metabolism and provides the precursors and cofactors required for synthesis of secondary metabolites. It is therefore pivotal to map the operating metabolic network in the central carbon metabolism in order to design metabolic engineering strategies towards construction of more efficient producers of specific metabolites. In this context, methods that allow rapid and reliable mapping of the central carbon metabolism are valuable. In the present study, a (13)C labelling-based method was used to identify the primary metabolic pathways of the poorly characterized antibiotic-producing actinomycete Nonomuraea sp. ATCC 39727. Surprisingly, it was found that Nonomuraea sp. ATCC 39272 predominantly metabolizes glucose via the Entner-Doudoroff (ED) pathway. This represents the first time that the ED pathway has been recognized as the main catabolic pathway in an actinomycete. The Nonomuraea genes encoding the key enzymes of the ED pathway were subsequently identified, sequenced and functionally described.     

L-arabinose metabolism in Herbaspirillum seropedicae.

The pathway for L-arabinose metabolism in Herbaspirillum seropedicae was shown to involve nonphosphorylated intermediates and to produce alpha-ketoglutarate. The activities of the enzymes and the natures of several intermediates were determined. The pathway was inducible by L-arabinose, and two key enzymes, L-arabinose dehydrogenase and 2-keto-glutarate semialdehyde dehydrogenase, were present in all strains of H. seropedicae tested.     

Characterization of the heterodimeric MegBIIa:MegBIIb aldo-keto reductase involved in the biosynthesis of L-mycarose from Micromonospora megalomicea

Two putative C3-ketoreductases, MegBIIa and MegBIIb (formerly MegBII and MegDVII, respectively), homologues to members of the family 12 of aldo-keto reductase (AKR12) superfamily of enzymes, were identified in the megalomicin gene cluster from Micromonospora megalomicea. Proteins from this family are involved in the metabolism of TDP-sugars by actinomycetes. MegBIIa was originally proposed to be involved in the l-mycarose biosynthetic pathway, while MegBIIb in the l-megosamine biosynthetic pathway. In this work we have investigated the role of these proteins in the biosynthesis of dTDP-l-mycarose. In vivo analysis of the dTDP-sugar intermediates indicated that neither MegBIIa nor its homologue, MegBIIb, was a fully active enzyme by itself. Surprisingly, C3-ketoreductase activity was observed only in the presence of both MegBIIa and MegBIIb, suggesting the formation of an active complex. Copurification and size exclusion chromatography experiments confirmed that MegBIIa and MegBIIb interact forming a 1:1 heterodimeric complex. Finally, a mycarose operon containing megBIIa and megBIIb together with the other biosynthetic genes of the l-mycarose pathway was constructed and tested by bioconversion experiments in Escherichia coli. High levels of mycarosyl-erythronolide B were produced under the condition tested, confirming the role of these two proteins in this metabolic pathway.     

Enzymes of serine biosynthesis in Rhodopseudomonas capsulata

Rhodopseudomonas capsulata has been shown to possess all the enzymatic activities of both the phosphorylated and nonphosphorylated pathways of serine biosynthesis. In addition there was an active serine hydroxymethyltransferase which catalyzed the reversible interconversion of serine and glycine. In cells grown photosynthetically with malate as the carbon source, the activities of the phosphorylated pathway enzymes were substantially higher than the analogous reactions of the nonphosphorylated sequence. l-Serine (1 mm) caused approximately 60%, inhibition of the first enzyme of the phosphorylated route, 3-phosphoglyceric acid dehydrogenase, but was less effective in inhibiting the last enzyme, phosphoserine phosphatase. Glycine also exerted a regulatory effect on this pathway but it was not as potent an inhibitor as serine. The inhibitions caused by serine and glycine were simply additive; there was no evidence of concerted feedback inhibition of the phosphorylated pathway by these amino acids.     

Vitamin B1 de novo synthesis in the human malaria parasite Plasmodium falciparum depends on external provision of 4-amino-5-hydroxymethyl-2-methylpyrimidine

Vitamin B1 (thiamine) is an essential cofactor for several key enzymes of carbohydrate metabolism. Mammals have to salvage this crucial nutrient from their diet to complement their deficiency of de novo synthesis. In contrast, bacteria, fungi, plants and, as reported here, Plasmodium falciparum, possess a vitamin B1 biosynthesis pathway. The plasmodial pathway identified consists of the three vitamin B1 biosynthetic enzymes 5-(2-hydroxy-ethyl)-4-methylthiazole (THZ) kinase (ThiM), 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP)/HMP-P kinase (ThiD) and thiamine phosphate synthase (ThiE). Recombinant PfThiM and PfThiD proteins were biochemically characterised, revealing K(m)app values of 68 microM for THZ and 12 microM for HMP. Furthermore, the ability of PfThiE for generating vitamin B1 was analysed by a complementation assay with thiE-negative E. coli mutants. All three enzymes are expressed throughout the developmental blood stages, as shown by Northern blotting, which indicates the presence of the vitamin B1 biosynthesis enzymes. However, cultivation of the parasite in minimal medium showed a dependency on the provision of HMP or thiamine. These results demonstrate that the human malaria parasite P. falciparum possesses active vitamin B1 biosynthesis, which depends on external provision of thiamine precursors.     

Carbon and sulfur metabolism in representatives of two clusters of bacteria of the genus Leucothrix: a comparative study

Major pathways of carbon and sulfur metabolisms were studied in representatives of two clusters of bacteria: Leucothrix thiophila (cluster I, strains 2WS, 4WS, and 6WS) and Leucothrix sp. (cluster II, strains 1WS, 3WS, and 5WS). All strains were capable of chemoorganoheterotrophic growth, as well as of chemolithoheterotrophic growth in the presence of reduced sulfur compounds. The bacteria were found to possess a complete set of the enzymes of the tricarboxylic acid cycle and glyoxylate cycle. The hydrogenase activity in cells of cluster I strains was an order of magnitude lower than in cluster II strains and in other known heterotrophic bacteria. Cells of bacteria of both clusters exhibited high activity levels of enzymes involved in the energy metabolism of sulfur. The oxidation of sulfur compounds and the operation of the electron-transport chain were shown to be related. Cluster II bacteria more efficiently use organic compounds in their energy metabolism, whereas cluster I bacteria are characterized by more efficient utilization of reduced sulfur compounds. During sulfite oxidation, cluster I bacteria can synthesize ATP both via substrate-level phosphorylation and oxidative phosphorylation, whereas cluster II bacteria synthesize ATP only via the latter process.