Feedback Inhibition of Deoxy-d-xylulose-5-phosphate Synthase Regulates the Methylerythritol 4-Phosphate Pathway

The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway leads to the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the precursors for isoprene and higher isoprenoids. Isoprene has significant effects on atmospheric chemistry, whereas other isoprenoids have diverse roles ranging from various biological processes to applications in commercial uses. Understanding the metabolic regulation of the MEP pathway is important considering the numerous applications of this pathway. The 1-deoxy-d-xylulose-5-phosphate synthase (DXS) enzyme was cloned from Populus trichocarpa, and the recombinant protein (PtDXS) was purified from Escherichia coli. The steady-state kinetic parameters were measured by a coupled enzyme assay. An LC-MS/MS-based assay involving the direct quantification of the end product of the enzymatic reaction, 1-deoxy-d-xylulose 5-phosphate (DXP), was developed. The effect of different metabolites of the MEP pathway on PtDXS activity was tested. PtDXS was inhibited by IDP and DMADP. Both of these metabolites compete with thiamine pyrophosphate for binding with the enzyme. An atomic structural model of PtDXS in complex with thiamine pyrophosphate and Mg²⁺ was built by homology modeling and refined by molecular dynamics simulations. The refined structure was used to model the binding of IDP and DMADP and indicated that IDP and DMADP might bind with the enzyme in a manner very similar to the binding of thiamine pyrophosphate. The feedback inhibition of PtDXS by IDP and DMADP constitutes an important mechanism of metabolic regulation of the MEP pathway and indicates that thiamine pyrophosphate-dependent enzymes may often be affected by IDP and DMADP.     

The Plastidial 2-C-Methyl-d-Erythritol 4-Phosphate Pathway Provides the Isoprenyl Moiety for Protein Geranylgeranylation in Tobacco BY-2 Cells

Protein farnesylation and geranylgeranylation are important posttranslational modifications in eukaryotic cells. We visualized in transformed Nicotiana tabacum Bright Yellow-2 (BY-2) cells the geranylgeranylation and plasma membrane localization of GFP-BD-CVIL, which consists of green fluorescent protein (GFP) fused to the C-terminal polybasic domain (BD) and CVIL isoprenylation motif from the Oryza sativa calmodulin, CaM61. Treatment with fosmidomycin (Fos) or oxoclomazone (OC), inhibitors of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, caused mislocalization of the protein to the nucleus, whereas treatment with mevinolin, an inhibitor of the cytosolic mevalonate pathway, did not. The nuclear localization of GFP-BD-CVIL in the presence of MEP pathway inhibitors was completely reversed by all-trans-geranylgeraniol (GGol). Furthermore, 1-deoxy-d-xylulose (DX) reversed the effects of OC, but not Fos, consistent with the hypothesis that OC blocks 1-deoxy-d-xylulose 5-phosphate synthesis, whereas Fos inhibits its conversion to 2-C-methyl-d-erythritol 4-phosphate. By contrast, GGol and DX did not rescue the nuclear mislocalization of GFP-BD-CVIL in the presence of a protein geranylgeranyltransferase type 1 inhibitor. Thus, the MEP pathway has an essential role in geranylgeranyl diphosphate (GGPP) biosynthesis and protein geranylgeranylation in BY-2 cells. GFP-BD-CVIL is a versatile tool for identifying pharmaceuticals and herbicides that interfere either with GGPP biosynthesis or with protein geranylgeranylation.     

Cloning and characterization of 2-C-methyl-D-erythritol-4-phosphate pathway genes for isoprenoid biosynthesis from Indian ginseng, Withania somnifera

Withania somnifera (L.) is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicines. Pharmaceutical activities of this herb are associated with presence of secondary metabolites known as withanolides, a class of phytosteroids synthesized via mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate pathways. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, not much is known about the genes responsible for biosynthesis of these compounds. In this study, we have characterized two genes encoding 1-deoxy-d-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7) and 1-deoxy-d-xylulose-5-phosphate reductase (DXR; EC 1.1.1.267) enzymes involved in the biosynthesis of isoprenoids. The full-length cDNAs of W. somnifera DXS (WsDXS) and DXR (WsDXR) of 2,154 and 1,428 bps encode polypeptides of 717 and 475 amino acids residues, respectively. The expression analysis suggests that WsDXS and WsDXR are differentially expressed in different tissues (with maximal expression in flower and young leaf), chemotypes of Withania, and in response to salicylic acid, methyl jasmonate, as well as in mechanical injury. Analysis of genomic organization of WsDXS shows close similarity with tomato DXS in terms of exon–intron arrangements. This is the first report on characterization of isoprenoid biosynthesis pathway genes from Withania.     

Characterization of 5-Chloro-5-Deoxy-d-Ribose 1-Dehydrogenase in Chloroethylmalonyl Coenzyme A Biosynthesis: SUBSTRATE AND REACTION PROFILING*

SalM is a short-chain dehydrogenase/reductase enzyme from the marine actinomycete Salinispora tropica that is involved in the biosynthesis of chloroethylmalonyl-CoA, a novel halogenated polyketide synthase extender unit of the proteasome inhibitor salinosporamide A. SalM was heterologously overexpressed in Escherichia coli and characterized in vitro for its substrate specificity, kinetics, and reaction profile. A sensitive real-time ¹³C NMR assay was developed to visualize the oxidation of 5-chloro-5-deoxy-d-ribose to 5-chloro-5-deoxy-d-ribono-γ-lactone in an NAD⁺-dependent reaction, followed by spontaneous lactone hydrolysis to 5-chloro-5-deoxy-d-ribonate. Although short-chain dehydrogenase/reductase enzymes are widely regarded as metal-independent, a strong divalent metal cation dependence for Mg²⁺, Ca²⁺, or Mn²⁺ was observed with SalM. Oxidative activity was also measured with the alternative substrates d-erythrose and d-ribose, making SalM the first reported stereospecific non-phosphorylative ribose 1-dehydrogenase.     

Pen and Pal Are Nucleotide-Sugar Dehydratases That Convert UDP-GlcNAc to UDP-6-Deoxy-d-GlcNAc-5,6-ene and Then to UDP-4-Keto-6-deoxy-l-AltNAc for CMP-Pseudaminic Acid Synthesis in Bacillus thuringiensis

CMP-pseudaminic acid is a precursor required for the O-glycosylation of flagellin in some pathogenic Gram-negative bacteria, a process known to be critical in bacterial motility and infection. However, little is known about flagellin glycosylation in Gram-positive bacteria. Here, we identified and functionally characterized an operon, named Bti_pse, in Bacillus thuringiensis israelensis ATCC 35646, which encodes seven different enzymes that together convert UDP-GlcNAc to CMP-pseudaminic acid. In contrast, Gram-negative bacteria complete this reaction with six enzymes. The first enzyme, which we named Pen, converts UDP-d-GlcNAc to an uncommon UDP-sugar, UDP-6-deoxy-d-GlcNAc-5,6-ene. Pen contains strongly bound NADP⁺ and has distinct UDP-GlcNAc 4-oxidase, 5,6-dehydratase, and 4-reductase activities. The second enzyme, which we named Pal, converts UDP-6-deoxy-d-GlcNAc-5,6-ene to UDP-4-keto-6-deoxy-l-AltNAc. Pal is NAD⁺-dependent and has distinct UDP-6-deoxy-d-GlcNAc-5,6-ene 4-oxidase, 5,6-reductase, and 5-epimerase activities. We also show here using NMR spectroscopy and mass spectrometry that in B. thuringiensis, the enzymatic product of Pen and Pal, UDP-4-keto-6-deoxy-l-AltNAc, is converted to CMP-pseudaminic acid by the sequential activities of a C4″-transaminase (Pam), a 4-N-acetyltransferase (Pdi), a UDP-hydrolase (Phy), an enzyme (Ppa) that adds phosphoenolpyruvate to form pseudaminic acid, and finally a cytidylyltransferase that condenses CTP to generate CMP-pseudaminic acid. Knowledge of the distinct dehydratase-like enzymes Pen and Pal and their role in CMP-pseudaminic acid biosynthesis in Gram-positive bacteria provides a foundation to investigate the role of pseudaminic acid and flagellin glycosylation in Bacillus and their involvement in bacterial motility and pathogenicity.     

Comparison of Predicted Epimerases and Reductases of the Campylobacter jejuni d-altro- and l-gluco-Heptose Synthesis Pathways

Uniquely modified heptoses found in surface carbohydrates of bacterial pathogens are potential therapeutic targets against such pathogens. Our recent biochemical characterization of the GDP-6-deoxy-d-manno- and GDP-6-deoxy-d-altro-heptose biosynthesis pathways has provided the foundation for elucidation of the more complex l-gluco-heptose synthesis pathway of Campylobacter jejuni strain NCTC 11168. In this work we use GDP-4-keto,6-deoxy-d-lyxo-heptose as a surrogate substrate to characterize three enzymes predicted to be involved in this pathway: WcaG_NCTC (also known as Cj1427), MlghB (Cj1430), and MlghC (Cj1428). We compare them with homologues involved in d-altro-heptose production: WcaG₈₁₁₇₆ (formerly WcaG), DdahB (Cjj1430), and DdahC (Cjj1427). We show that despite high levels of similarity, the enzymes have pathway-specific catalytic activities and substrate specificities. MlghB forms three products via C3 and C5 epimerization activities, whereas its DdahB homologue only had C3 epimerase activity along its cognate pathway. MlghC is specific for the double C3/C5 epimer generated by MlghB and produces l-gluco-heptose via stereospecific C4 reductase activity. In contrast, its homologue DdahC only uses the C3 epimer to yield d-altro-heptose via C4 reduction. Finally, we show that WcaG_NCTC is not necessary for l-gluco-heptose synthesis and does not affect its production by MlghB and MlghC, in contrast to its homologue WcaG₈₁₁₇₆, that has regulatory activity on d-altro-heptose synthesis. These studies expand our fundamental understanding of heptose modification, provide new glycobiology tools to synthesize novel heptose derivatives with biomedical applications, and provide a foundation for the structure function analysis of these enzymes.     

Discovery of β-1,4-d-Mannosyl-N-acetyl-d-glucosamine Phosphorylase Involved in the Metabolism of N-Glycans

A gene cluster involved in N-glycan metabolism was identified in the genome of Bacteroides thetaiotaomicron VPI-5482. This gene cluster encodes a major facilitator superfamily transporter, a starch utilization system-like transporter consisting of a TonB-dependent oligosaccharide transporter and an outer membrane lipoprotein, four glycoside hydrolases (α-mannosidase, β-N-acetylhexosaminidase, exo-α-sialidase, and endo-β-N-acetylglucosaminidase), and a phosphorylase (BT1033) with unknown function. It was demonstrated that BT1033 catalyzed the reversible phosphorolysis of β-1,4-d-mannosyl-N-acetyl-d-glucosamine in a typical sequential Bi Bi mechanism. These results indicate that BT1033 plays a crucial role as a key enzyme in the N-glycan catabolism where β-1,4-d-mannosyl-N-acetyl-d-glucosamine is liberated from N-glycans by sequential glycoside hydrolase-catalyzed reactions, transported into the cell, and intracellularly converted into α-d-mannose 1-phosphate and N-acetyl-d-glucosamine. In addition, intestinal anaerobic bacteria such as Bacteroides fragilis, Bacteroides helcogenes, Bacteroides salanitronis, Bacteroides vulgatus, Prevotella denticola, Prevotella dentalis, Prevotella melaninogenica, Parabacteroides distasonis, and Alistipes finegoldii were also suggested to possess the similar metabolic pathway for N-glycans. A notable feature of the new metabolic pathway for N-glycans is the more efficient use of ATP-stored energy, in comparison with the conventional pathway where β-mannosidase and ATP-dependent hexokinase participate, because it is possible to directly phosphorylate the d-mannose residue of β-1,4-d-mannosyl-N-acetyl-d-glucosamine to enter glycolysis. This is the first report of a metabolic pathway for N-glycans that includes a phosphorylase. We propose 4-O-β-d-mannopyranosyl-N-acetyl-d-glucosamine:phosphate α-d-mannosyltransferase as the systematic name and β-1,4-d-mannosyl-N-acetyl-d-glucosamine phosphorylase as the short name for BT1033.     

Accumulation of d-Glucose from Pentoses by Metabolically Engineered Escherichia coli

Escherichia coli that is unable to metabolize d-glucose (with knockouts in ptsG, manZ, and glk) accumulates a small amount of d-glucose (yield of about 0.01 g/g) during growth on the pentoses d-xylose or l-arabinose as a sole carbon source. Additional knockouts in the zwf and pfkA genes, encoding, respectively, d-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (E. coli MEC143), increased accumulation to greater than 1 g/liter d-glucose and 100 mg/liter d-mannose from 5 g/liter d-xylose or l-arabinose. Knockouts of other genes associated with interconversions of d-glucose-phosphates demonstrate that d-glucose is formed primarily by the dephosphorylation of d-glucose-6-phosphate. Under controlled batch conditions with 20 g/liter d-xylose, MEC143 generated 4.4 g/liter d-glucose and 0.6 g/liter d-mannose. The results establish a direct link between pentoses and hexoses and provide a novel strategy to increase carbon backbone length from five to six carbons by directing flux through the pentose phosphate pathway.     

Probing the Sophisticated Synergistic Allosteric Regulation of Aromatic Amino Acid Biosynthesis in Mycobacterium tuberculosis Using ?-Amino Acids

Chirality plays a major role in recognition and interaction of biologically important molecules. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of the shikimate pathway, which is responsible for the synthesis of aromatic amino acids in bacteria and plants, and a potential target for the development of antibiotics and herbicides. DAH7PS from Mycobacterium tuberculosis (MtuDAH7PS) displays an unprecedented complexity of allosteric regulation, with three interdependent allosteric binding sites and a ternary allosteric response to combinations of the aromatic amino acids l-Trp, l-Phe and l-Tyr. In order to further investigate the intricacies of this system and identify key residues in the allosteric network of MtuDAH7PS, we studied the interaction of MtuDAH7PS with aromatic amino acids that bear the non-natural d-configuration, and showed that the d-amino acids do not elicit an allosteric response. We investigated the binding mode of d-amino acids using X-ray crystallography, site directed mutagenesis and isothermal titration calorimetry. Key differences in the binding mode were identified: in the Phe site, a hydrogen bond between the amino group of the allosteric ligands to the side chain of Asn175 is not established due to the inverted configuration of the ligands. In the Trp site, d-Trp forms no interaction with the main chain carbonyl group of Thr240 and less favourable interactions with Asn237 when compared to the l-Trp binding mode. Investigation of the MtuDAH7PS_N175A variant further supports the hypothesis that the lack of key interactions in the binding mode of the aromatic d-amino acids are responsible for the absence of an allosteric response, which gives further insight into which residues of MtuDAH7PS play a key role in the transduction of the allosteric signal.     

Metabolic Mechanism of Mannan in a Ruminal Bacterium,Ruminococcus albus,Involving Two Mannoside Phosphorylases and Cellobiose 2-Epimerase: DISCOVERY OF A NEW CARBOHYDRATE PHOSPHORYLASE, β-1,4-MANNOOLIGOSACCHARIDE PHOSPHORYLASE*

Ruminococcus albus is a typical ruminal bacterium digesting cellulose and hemicellulose. Cellobiose 2-epimerase (CE; EC 5.1.3.11), which converts cellobiose to 4-O-β-d-glucosyl-d-mannose, is a particularly unique enzyme in R. albus, but its physiological function is unclear. Recently, a new metabolic pathway of mannan involving CE was postulated for another CE-producing bacterium, Bacteroides fragilis. In this pathway, β-1,4-mannobiose is epimerized to 4-O-β-d-mannosyl-d-glucose (Man-Glc) by CE, and Man-Glc is phosphorolyzed to α-d-mannosyl 1-phosphate (Man1P) and d-glucose by Man-Glc phosphorylase (MP; EC 2.4.1.281). Ruminococcus albus NE1 showed intracellular MP activity, and two MP isozymes, RaMP1 and RaMP2, were obtained from the cell-free extract. These enzymes were highly specific for the mannosyl residue at the non-reducing end of the substrate and catalyzed the phosphorolysis and synthesis of Man-Glc through a sequential Bi Bi mechanism. In a synthetic reaction, RaMP1 showed high activity only toward d-glucose and 6-deoxy-d-glucose in the presence of Man1P, whereas RaMP2 showed acceptor specificity significantly different from RaMP1. RaMP2 acted on d-glucose derivatives at the C2- and C3-positions, including deoxy- and deoxyfluoro-analogues and epimers, but not on those substituted at the C6-position. Furthermore, RaMP2 had high synthetic activity toward the following oligosaccharides: β-linked glucobioses, maltose, N,N′-diacetylchitobiose, and β-1,4-mannooligosaccharides. Particularly, β-1,4-mannooligosaccharides served as significantly better acceptor substrates for RaMP2 than d-glucose. In the phosphorolytic reactions, RaMP2 had weak activity toward β-1,4-mannobiose but efficiently degraded β-1,4-mannooligosaccharides longer than β-1,4-mannobiose. Consequently, RaMP2 is thought to catalyze the phosphorolysis of β-1,4-mannooligosaccharides longer than β-1,4-mannobiose to produce Man1P and β-1,4-mannobiose.     

Effect of down-regulating 1-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-xylulose-5-phosphate reductoisomerase by RNAi on growth and artemisinin biosynthesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), an important enzyme in the 2-c-methyl-d-erythritol-4-phosphate (MEP) pathway in plant plastids, provides the basic five-carbon units for isoprenoid biosynthesis. To investigate the roles of the MEP pathway in regulating growth, development and artemisinin biosynthesis of Artemisia annua L., we used RNA interference technology to generate transgenic plants with suppressed expression of DXR in A. annua (AaDXR). Suppression of AaDXR resulted in shorter stems, decreased branch numbers and leaf area, lower density of leaf trichomes. Although AaDXR-RNAi plants had no significant changes on the stomatal conductance, the net photosynthesis rate was decreased by 20.0–31.4% due to the marked decline in the contents of chlorophyll. Decreased levels of endogenous gibberellic acid (GA₃) and abscisic acid were also detected in the transgenic lines. The artemisinin contents in leaves of all tested transgenic lines declined by 41.8–73.4% at the vegetative stage and 61.5–63.6% at the stages of flowering. The enhancement of artemisinin contents by methyl jasmonate at 300 µM has been abolished at seedling and vegetative stages in AaDXR-RNAi plants. These results demonstrate that AaDXR play import roles in the control of plan vegetative growth and artemisinin biosynthesis in A. annua.     

Crystal structure of a zinc-dependent D-serine dehydratase from chicken kidney

d-Serine is a physiological co-agonist of the N-methyl-d-aspartate receptor. It regulates excitatory neurotransmission, which is important for higher brain functions in vertebrates. In mammalian brains, d-amino acid oxidase degrades d-serine. However, we have found recently that in chicken brains the oxidase is not expressed and instead a d-serine dehydratase degrades d-serine. The primary structure of the enzyme shows significant similarities to those of metal-activated d-threonine aldolases, which are fold-type III pyridoxal 5′-phosphate (PLP)-dependent enzymes, suggesting that it is a novel class of d-serine dehydratase. In the present study, we characterized the chicken enzyme biochemically and also by x-ray crystallography. The enzyme activity on d-serine decreased 20-fold by EDTA treatment and recovered nearly completely by the addition of Zn²⁺. None of the reaction products that would be expected from side reactions of the PLP-d-serine Schiff base were detected during the >6000 catalytic cycles of dehydration, indicating high reaction specificity. We have determined the first crystal structure of the d-serine dehydratase at 1.9 Å resolution. In the active site pocket, a zinc ion that coordinates His³⁴⁷ and Cys³⁴⁹ is located near the PLP-Lys⁴⁵ Schiff base. A theoretical model of the enzyme-d-serine complex suggested that the hydroxyl group of d-serine directly coordinates the zinc ion, and that the ϵ-NH₂ group of Lys⁴⁵ is a short distance from the substrate Cα atom. The α-proton abstraction from d-serine by Lys⁴⁵ and the elimination of the hydroxyl group seem to occur with the assistance of the zinc ion, resulting in the strict reaction specificity.     

d-Xylose Degradation Pathway in the Halophilic Archaeon Haloferax volcanii

The pathway of d-xylose degradation in archaea is unknown. In a previous study we identified in Haloarcula marismortui the first enzyme of xylose degradation, an inducible xylose dehydrogenase (Johnsen, U., and Schönheit, P. (2004) J. Bacteriol. 186, 6198–6207). Here we report a comprehensive study of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. The analyses include the following: (i) identification of the degradation pathway in vivo following ¹³C-labeling patterns of proteinogenic amino acids after growth on [¹³C]xylose; (ii) identification of xylose-induced genes by DNA microarray experiments; (iii) characterization of enzymes; and (iv) construction of in-frame deletion mutants and their functional analyses in growth experiments. Together, the data indicate that d-xylose is oxidized exclusively to the tricarboxylic acid cycle intermediate α-ketoglutarate, involving d-xylose dehydrogenase (HVO_B0028), a novel xylonate dehydratase (HVO_B0038A), 2-keto-3-deoxyxylonate dehydratase (HVO_B0027), and α-ketoglutarate semialdehyde dehydrogenase (HVO_B0039). The functional involvement of these enzymes in xylose degradation was proven by growth studies of the corresponding in-frame deletion mutants, which all lost the ability to grow on d-xylose, but growth on glucose was not significantly affected. This is the first report of an archaeal d-xylose degradation pathway that differs from the classical d-xylose pathway in most bacteria involving the formation of xylulose 5-phosphate as an intermediate. However, the pathway shows similarities to proposed oxidative pentose degradation pathways to α-ketoglutarate in few bacteria, e.g. Azospirillum brasilense and Caulobacter crescentus, and in the archaeon Sulfolobus solfataricus.d-Xylose, a constituent of the polymer xylan, is the major component of the hemicellulose plant cell wall material and thus one of the most abundant carbohydrates in nature. The utilization of d-xylose by microorganisms has been described in detail in bacteria and fungi, for which two different catabolic pathways have been reported. In many bacteria, such as Escherichia coli, Bacillus, and Lactobacillus species, xylose is converted by the activities of xylose isomerase and xylulose kinase to xylulose 5-phosphate as an intermediate, which is further degraded mainly by the pentose phosphate cycle or phosphoketolase pathway. Most fungi convert xylose to xylulose 5-phosphate via xylose reductase, xylitol dehydrogenase, and xylulose kinase. Xylulose 5-phosphate is also an intermediate of the most common l-arabinose degradation pathway in bacteria, e.g. of E. coli, via activities of isomerase, kinase, and epimerase (1).Recently, by genetic evidence, a third pathway of xylose degradation was proposed for the bacterium Caulobacter crescentus, in analogy to an alternative catabolic pathway of l-arabinose, reported for some bacteria, including species of Azospirillum, Pseudomonas, Rhizobium, Burkholderia, and Herbasprillum (2, 3). In these organisms l-arabinose is oxidatively degraded to α-ketoglutarate, an intermediate of the tricarboxylic acid cycle, via the activities of l-arabinose dehydrogenase, l-arabinolactonase, and two successive dehydration reactions forming 2-keto-3-deoxy-l-arabinoate and α-ketoglutarate semialdehyde; the latter compound is further oxidized to α-ketoglutarate via NADP⁺-specific α-ketoglutarate semialdehyde dehydrogenase (KGSADH).² In a few Pseudomonas and Rhizobium species, a variant of this l-arabinose pathway was described involving aldolase cleavage of the intermediate 2-keto-3-deoxy-l-arabinoate to pyruvate and glycolaldehyde, rather than its dehydration and oxidation to α-ketoglutarate (4). Because of the presence of some analogous enzyme activities in xylose-grown cells of Azosprillum and Rhizobium, the oxidative pathway and its variant was also proposed as a catabolic pathway for d-xylose. Recent genetic analysis of Caulobacter crecentus indicates the presence of an oxidative pathway for d-xylose degradation to α-ketoglutarate. All genes encoding xylose dehydrogenase and putative lactonase, xylonate dehydratase, 2-keto-3-deoxylonate dehydratase, and KGSADH were found to be located on a xylose-inducible operon (5). With exception of xylose dehydrogenase, which has been partially characterized, the other postulated enzymes of the pathway have not been biochemically analyzed.The pathway of d-xylose degradation in the domain of archaea has not been studied so far. First analyses with the halophilic archaeon Haloarcula marismortui indicate that the initial step of d-xylose degradation involves a xylose-inducible xylose dehydrogenase (6) suggesting an oxidative pathway of xylose degradation to α-ketoglutarate, or to pyruvate and glycolaldehyde, in analogy to the proposed oxidative bacterial pentose degradation pathways. Recently, a detailed study of d-arabinose catabolism in the thermoacidophilic crenarchaeon Sulfolobus solfataricus was reported. d-Arabinose was found to be oxidized to α-ketoglutarate involving d-arabinose dehydrogenase, d-arabinoate dehydratase, 2-keto-3-deoxy-d-arabinoate dehydratase, and α-ketoglutarate semialdehyde dehydrogenase (3).In this study, we present a comprehensive analysis of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. This halophilic archaeon was chosen because it exerts several suitable properties for the analyses. For example, it can be cultivated on synthetic media with sugars, e.g. xylose, an advantage for in vivo labeling studies in growing cultures. Furthermore, a shotgun DNA microarray of H. volcanii is available (7) allowing the identification of xylose-inducible genes, and H. volcanii is one of the few archaea for which an efficient protocol was recently described to generate in-frame deletion mutants.Accordingly, the d-xylose degradation pathway was elucidated following in vivo labeling experiments with [¹³C]xylose, DNA microarray analyses, and the characterization of enzymes involved and their encoding genes. The functional involvement of genes and enzymes was proven by constructing corresponding in-frame deletion mutants and their analysis by selective growth experiments on xylose versus glucose. The data show that d-xylose was exclusively degraded to α-ketoglutarate involving xylose dehydrogenase, a novel xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase, and α-ketoglutarate semialdehyde dehydrogenase.     

An l-glucose Catabolic Pathway in Paracoccus Species 43P

An l-glucose-utilizing bacterium, Paracoccus sp. 43P, was isolated from soil by enrichment cultivation in a minimal medium containing l-glucose as the sole carbon source. In cell-free extracts from this bacterium, NAD⁺-dependent l-glucose dehydrogenase was detected as having sole activity toward l-glucose. This enzyme, LgdA, was purified, and the lgdA gene was found to be located in a cluster of putative inositol catabolic genes. LgdA showed similar dehydrogenase activity toward scyllo- and myo-inositols. l-Gluconate dehydrogenase activity was also detected in cell-free extracts, which represents the reaction product of LgdA activity toward l-glucose. Enzyme purification and gene cloning revealed that the corresponding gene resides in a nine-gene cluster, the lgn cluster, which may participate in aldonate incorporation and assimilation. Kinetic and reaction product analysis of each gene product in the cluster indicated that they sequentially metabolize l-gluconate to glycolytic intermediates, d-glyceraldehyde-3-phosphate, and pyruvate through reactions of C-5 epimerization by dehydrogenase/reductase, dehydration, phosphorylation, and aldolase reaction, using a pathway similar to l-galactonate catabolism in Escherichia coli. Gene disruption studies indicated that the identified genes are responsible for l-glucose catabolism.     

Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.     

Identification and Characterization of d-Hydroxyproline Dehydrogenase and Δ1-Pyrroline-4-hydroxy-2-carboxylate Deaminase Involved in Novel l-Hydroxyproline Metabolism of Bacteria: METABOLIC CONVERGENT EVOLUTION*

l-Hydroxyproline (4-hydroxyproline) mainly exists in collagen, and most bacteria cannot metabolize this hydroxyamino acid. Pseudomonas putida and Pseudomonas aeruginosa convert l-hydroxyproline to α-ketoglutarate via four hypothetical enzymatic steps different from known mammalian pathways, but the molecular background is rather unclear. Here, we identified and characterized for the first time two novel enzymes, d-hydroxyproline dehydrogenase and Δ¹-pyrroline-4-hydroxy-2-carboxylate (Pyr4H2C) deaminase, involved in this hypothetical pathway. These genes were clustered together with genes encoding other catalytic enzymes on the bacterial genomes. d-Hydroxyproline dehydrogenases from P. putida and P. aeruginosa were completely different from known bacterial proline dehydrogenases and showed similar high specificity for substrate (d-hydroxyproline) and some artificial electron acceptor(s). On the other hand, the former is a homomeric enzyme only containing FAD as a prosthetic group, whereas the latter is a novel heterododecameric structure consisting of three different subunits (α₄β₄γ₄), and two FADs, FMN, and [2Fe-2S] iron-sulfur cluster were contained in αβγ of the heterotrimeric unit. These results suggested that the l-hydroxyproline pathway clearly evolved convergently in P. putida and P. aeruginosa. Pyr4H2C deaminase is a unique member of the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein family, and its activity was competitively inhibited by pyruvate, a common substrate for other dihydrodipicolinate synthase/N-acetylneuraminate lyase proteins. Furthermore, disruption of Pyr4H2C deaminase genes led to loss of growth on l-hydroxyproline (as well as d-hydroxyproline) but not l- and d-proline, indicating that this pathway is related only to l-hydroxyproline degradation, which is not linked to proline metabolism.     

Potent inhibitors of a shikimate pathway enzyme from Mycobacterium tuberculosis: combining mechanism- and modeling-based design

Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-d-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 Å. Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined.     

Sugar transport across the peritubular face of renal cells of the flounder

The transport of some sugars at the antiluminal face of renal cells was studied using teased tubules of flounder (Pseudopleuronectes americanus). The analytical procedure allowed the determination of both free and total (free plus phosphorylated) tissue sugars. The inulin space of the preparation was 0.333 ± 0.017 kg/kg wet wt (7 animals, 33 analyses). The nonmetabolizable α-methyl-D-glucoside entered the cells by a carrier-mediated (phloridzin-sensitive), ouabain-insensitive process. The steady-state tissue/medium ratio was systematically below that for diffusion equilibrium. D-Glucose was a poor inhibitor of α-methyl-glucoside transport, D-galactose was ineffective. The phloridzin-sensitive transport processes of 2-deoxy-D-glucose,D-galactose,and 2-deoxy-D-galactose were associated with considerable phosphorylation. Kinetic evidence suggested that these sugars were transported in free form and subsequently were phosphorylated. 2-Deoxy-D-glucose accumulated in the cells against a slight concentration gradient. This transport was greatly inhibited by D-glucose, whereas α-methyl-glucoside and also D-galactose and its 2-deoxy-derivative were ineffective. D-Galactose and 2-deoxy-D-galactose mutually competed for transport; D-glucose, 2-deoxy-D-glucose, and α-methyl-D-glucoside were ineffective. Studies using various sugars as inhibitors suggest the presence of three carrier-mediated pathways of sugar transport at the antiluminal cell face of the flounder renal tubule: the pathway of α-methyl-D-glucoside (not shared by D-glucose); the pathway commonly shared by 2-deoxy-D-glucose and D-glucose; the pathway shared by D-galactose and 2-deoxy-D-galactose.