Dihydroxyacid Dehydratase Research Articles

The genetic architecture of branched-chain amino acid accumulation in tomato fruits

Previous studies of the genetic architecture of fruit metabolic composition have allowed us to identify four strongly conserved co-ordinate quantitative trait loci (QTL) for the branched-chain amino acids (BCAAs). This study has been extended here to encompass the other 23 enzymes described to be involved in the pathways of BCAA synthesis and degradation. On coarse mapping the chromosomal location of these enzymes, it was possible to define the map position of 24 genes. Of these genes eight co-localized, or mapped close to BCAA QTL including those encoding ketol-acid reductoisomerase (KARI), dihydroxy-acid dehydratase (DHAD), and isopropylmalate dehydratase (IPMD). Quantitative evaluation of the expression levels of these genes revealed that the S. pennellii allele of IPMD demonstrated changes in the expression level of this gene, whereas those of KARI and DHAD were invariant across the genotypes. Whilst the antisense inhibition of IPMD resulted in increased BCAA, the antisense inhibition of neither KARI nor DHAD produced a clear effect in fruit BCAA contents. The results are discussed both with respect to the roles of these specific enzymes within plant amino acid metabolism and within the context of current understanding of the regulation of plant branched-chain amino acid metabolism.

Escherichia coli W as a new platform strain for the enhanced production of L‐Valine by systems metabolic engineering

A less frequently employed Escherichia coli strain W, yet possessing useful metabolic characteristics such as less acetic acid production and high L-valine tolerance, was metabolically engineered for the production of L-valine. The ilvA gene was deleted to make more pyruvate, a key precursor for L-valine, available for enhanced L-valine biosynthesis. The lacI gene was deleted to allow constitutive expression of genes under the tac or trc promoter. The ilvBN(mut) genes encoding feedback-resistant acetohydroxy acid synthase (AHAS) I and the L-valine biosynthetic ilvCED genes encoding acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and branched chain amino acid aminotransferase, respectively, were amplified by plasmid-based overexpression. The global regulator Lrp and L-valine exporter YgaZH were also amplified by plasmid-based overexpression. The engineered E. coli W (ΔlacI ΔilvA) strain overexpressing the ilvBN(mut) , ilvCED, ygaZH, and lrp genes was able to produce an impressively high concentration of 60.7 g/L L-valine by fed-batch culture in 29.5 h, resulting in a high volumetric productivity of 2.06 g/L/h. The most notable finding is that there was no other byproduct produced during L-valine production. The results obtained in this study suggest that E. coli W can be a good alternative to Corynebacterium glutamicum and E. coli K-12, which have so far been the most efficient L-valine producer. Furthermore, it is expected that various bioproducts including other amino acids might be more efficiently produced by this revisited platform strain of E. coli.

The Penicillium Chrysogenum Extracellular Proteome. Conversion from a Food-rotting Strain to a Versatile Cell Factory for White Biotechnology

The filamentous fungus Penicillium chrysogenum is well-known by its ability to synthesize β-lactam antibiotics as well as other secondary metabolites. Like other filamentous fungi, this microorganism is an excellent host for secretion of extracellular proteins because of the high capacity of its protein secretion machinery. In this work, we have characterized the extracellular proteome reference map of P. chrysogenum Wisconsin 54-1255 by two-dimensional gel electrophoresis. This method allowed the correct identification of 279 spots by peptide mass fingerprinting and tandem MS. These 279 spots included 328 correctly identified proteins, which corresponded to 131 different proteins and their isoforms. One hundred and two proteins out of 131 were predicted to contain either classical or nonclassical secretion signal peptide sequences, providing evidence of the authentic extracellular location of these proteins. Proteins with higher representation in the extracellular proteome were those involved in plant cell wall degradation (polygalacturonase, pectate lyase, and glucan 1,3-β-glucosidase), utilization of nutrients (extracellular acid phosphatases and 6-hydroxy-d-nicotine oxidase), and stress response (catalase R). This filamentous fungus also secretes enzymes specially relevant for food industry, such as sulfydryl oxidase, dihydroxy-acid dehydratase, or glucoamylase. The identification of several antigens in the extracellular proteome also highlights the importance of this microorganism as one of the main indoor allergens. Comparison of the extracellular proteome among three strains of P. chrysogenum, the wild-type NRRL 1951, the Wis 54-1255 (an improved, moderate penicillin producer), and the AS-P-78 (a penicillin high-producer), provided important insights to consider improved strains of this filamentous fungus as versatile cell-factories of interest, beyond antibiotic production, for other aspects of white biotechnology.

Metabolic Engineering of Corynebacterium glutamicum for 2-Ketoisovalerate Production

2-Ketoisovalerate is used as a therapeutic agent, and a 2-ketoisovalerate-producing organism may serve as a platform for products deriving from this 2-keto acid. We engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of 2-ketoisovalerate from glucose by deletion of the aceE gene encoding the E1p subunit of the pyruvate dehydrogenase complex, deletion of the transaminase B gene ilvE, and additional overexpression of the ilvBNCD genes, encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase (AHAS), acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. 2-Ketoisovalerate production was further improved by deletion of the pyruvate:quinone oxidoreductase gene pqo. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 188 ± 28 mM (21.8 ± 3.2 g liter(-1)) 2-ketoisovalerate and showed a product yield of about 0.47 ± 0.05 mol per mol (0.3 ± 0.03 g per g) of glucose and a volumetric productivity of about 4.6 ± 0.6 mM (0.53 ± 0.07 g liter(-1)) 2-ketoisovalerate per h in the overall production phase. In studying the influence of the three branched-chain 2-keto acids 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate on the AHAS activity, we observed a competitive inhibition of the AHAS enzyme by 2-ketoisovalerate.

Downregulation of Rv0189c, encoding a dihydroxyacid dehydratase, affects growth of Mycobacterium tuberculosis in vitro and in mice

Dihydroxyacid dehydratase (DHAD), a key enzyme involved in branched-chain amino acid (BCAA) biosynthesis, catalyses the synthesis of 2-ketoacids from dihydroxyacids. In Mycobacterium tuberculosis, DHAD is encoded by gene Rv0189c, and it shares 40% amino acid sequence identity and conserved motifs with DHAD of Escherichia coli encoded by ilvD. In this study, Rv0189c was overexpressed in E. coli and the resultant protein was characterized as a homodimer (~155 kDa). Functional characterization of Rv0189c was established by biochemical testing and by genetic complementation of an intron-disrupted ilvD-auxotrophic mutant of E. coli to prototrophy. Growth of M. tuberculosis, E. coli BL21(DE3) and recombinant E. coli BL21(DE3) ΔilvD carrying Rv0189c was inhibited by transient nitric oxide (NO) exposure in minimal medium but growth was restored if the medium was supplemented with BCAA (isoleucine, leucine and valine). This suggested that inactivation of Rv0189c by NO probably inhibited bacterial growth. The role of Rv0189c in M. tuberculosis was elucidated by antisense and sense RNA constructs. Growth of M. tuberculosis transformed with a plasmid encoding antisense mRNA was markedly poor in the lungs of infected mice and in Middlebrook 7H9 broth compared to that of sense and vector-alone transformants, but growth was normal when the medium was supplemented with BCAA. Upregulation of Rv0189c was observed during the early exponential phase of growth, under acid stress and ex vivo, suggesting that Rv0189c has a role in the survival of M. tuberculosis during normal and stress conditions. It may be concluded that the DHAD encoded by Rv0189c is essential for the survival of M. tuberculosis and could be a potential drug/vaccine target, as it is absent in mammals.

Factors enhancing l-valine production by the growth-limited l-isoleucine auxotrophic strain Corynebacterium glutamicum ΔilvA ΔpanB ilvNM13 (pECKAilvBNC)

Cell growth limitation is known to be an important condition that enhances L: -valine synthesis in Corynebacterium glutamicum recombinant strains with L: -isoleucine auxotrophy. To identify whether it is the limited availability of L: -isoleucine itself or the L: -isoleucine limitation-induced rel-dependent ppGpp-mediated stringent response that is essential for the enhancement of L: -valine synthesis in growth-limited C. glutamicum cells, we deleted the rel gene, thereby constructing a relaxed (rel (-) ) C. glutamicum DeltailvA DeltapanB Deltarel ilvNM13 (pECKAilvBNC) strain. Variations in enzyme activity and L: -valine synthesis in rel (+) and rel (-) strains under conditions of L: -isoleucine excess and limitation were investigated. A sharp increase in acetohydroxy acid synthase (AHAS) activity, a slight increase in acetohydroxyacid isomeroreductase (AHAIR) activity, and a dramatic increase in L: -valine synthesis were observed in both rel (+) and rel (-) cells exposed to L: -isoleucine limitation. Although the positive effect of induction of the stringent response on AHAS and AHAIR upregulation in cells was not confirmed, we found the stringent response to be beneficial for maintaining increased AHAS, dihydroxyacid dehydratase, and transaminase B activity and L: -valine synthesis in cells during the stationary growth phase.

IscA/SufA paralogues are required for the [4Fe-4S] cluster assembly in enzymes of multiple physiological pathways inEscherichia coliunder aerobic growth conditions

IscA/SufA paralogues are the members of the iron-sulfur cluster assembly machinery in Escherichia coli. Whereas deletion of either IscA or SufA has only a mild effect on cell growth, deletion of both IscA and SufA results in a null-growth phenotype in minimal medium under aerobic growth conditions. Here we report that cell growth of the iscA/sufA double mutant (E. coli strain in which both iscA and sufA had been in-frame-deleted) can be partially restored by supplementing with BCAAs (branched-chain amino acids) and thiamin. We further demonstrate that deletion of IscA/SufA paralogues blocks the [4Fe-4S] cluster assembly in IlvD (dihydroxyacid dehydratase) of the BCAA biosynthetic pathway in E. coli cells under aerobic conditions and that addition of the iron-bound IscA/SufA efficiently promotes the [4Fe-4S] cluster assembly in IlvD and restores the enzyme activity in vitro, suggesting that IscA/SufA may act as an iron donor for the [4Fe-4S] cluster assembly under aerobic conditions. Additional studies reveal that IscA/SufA are also required for the [4Fe-4S] cluster assembly in enzyme ThiC of the thiamin-biosynthetic pathway, aconitase B of the citrate acid cycle and endonuclease III of the DNA-base-excision-repair pathway in E. coli under aerobic conditions. Nevertheless, deletion of IscA/SufA does not significantly affect the [2Fe-2S] cluster assembly in the redox transcription factor SoxR, ferredoxin and the siderophore-iron reductase FhuF. The results suggest that the biogenesis of the [4Fe-4S] clusters and the [2Fe-2S] clusters may have distinct pathways and that IscA/SufA paralogues are essential for the [4Fe-4S] cluster assembly, but are dispensable for the [2Fe-2S] cluster assembly in E. coli under aerobic conditions.

Role of iron‐sulfur cluster in E. coli DinG helicase

The E. coli DinG protein encoded by the DNA damage‐inducible gene (dinG) is a member of the helicase superfamily 2. It has the DNA‐dependent ATPase activity and ATP‐dependent helicase activity. In this study, we report that the purified DinG protein contains a [4Fe‐4S] cluster with a midpoint redox potential ‐390±23 mV (pH 8.0). More importantly, the DinG helicase activity is inactivated when its [4Fe‐4S] is reduced and reactivated when the cluster is re‐oxidized, suggesting that the redox state of the [4Fe‐4S] cluster in DinG can modulate helicase activity. Unlike the [4F‐4S] cluster of E. coli dihydroxyacid dehydratase, the [4Fe‐4S] cluster in DinG is resistant to the oxidative stress caused by hydrogen peroxide and remains active. On the other hand, the DinG helicase activity is completely abolished by nitric oxide (NO) through modification of its 4Fe‐4S cluster and almost fully restored when the NO‐modified [4Fe‐4S] cluster is reassembled. The results suggest that the [4Fe‐4S] cluster in DinG may act as a sensor of the cellular redox potential and that the NO modification of the iron‐sulfur cluster in DinG and other DNA repair enzymes may contribute to the NO‐mediated genomic instability.

Redox Control of the DNA Damage-inducible Protein DinG Helicase Activity via Its Iron-Sulfur Cluster

The Escherichia coli DNA damage-inducible protein DinG, a member of the superfamily 2 DNA helicases, has been implicated in the nucleotide excision repair and recombinational DNA repair pathways. Combining UV-visible absorption, EPR, and enzyme activity measurements, we demonstrate here that E. coli DinG contains a redox-active [4Fe-4S] cluster with a midpoint redox potential (E(m)) of -390 +/- 23 mV (pH 8.0) and that reduction of the [4Fe-4S] cluster reversibly switches off the DinG helicase activity. Unlike the [4Fe-4S] cluster in E. coli dihydroxyacid dehydratase, the DinG [4Fe-4S] cluster is stable, and the enzyme remains fully active after exposure to 100-fold excess of hydrogen peroxide, indicating that DinG could be functional under oxidative stress conditions. However, the DinG [4Fe-4S] cluster can be efficiently modified by nitric oxide (NO), forming the DinG-bound dinitrosyl iron complex with the concomitant inactivation of helicase activity in vitro and in vivo. Reassembly of the [4Fe-4S] cluster in NO-modified DinG restores helicase activity, indicating that the iron-sulfur cluster in DinG is the primary target of NO cytotoxicity. The results led us to propose that the iron-sulfur cluster in DinG may act as a sensor of intracellular redox potential to modulate its helicase activity and that modification of the iron-sulfur cluster in DinG and likely in other DNA repair enzymes by NO may contribute to NO-mediated genomic instability.

Reactivity of nitric oxide with the [4Fe–4S] cluster of dihydroxyacid dehydratase fromEscherichia coli

Although the NO (nitric oxide)-mediated modification of iron-sulfur proteins has been well-documented in bacteria and mammalian cells, specific reactivity of NO with iron-sulfur proteins still remains elusive. In the present study, we report the first kinetic characterization of the reaction between NO and iron-sulfur clusters in protein using the Escherichia coli IlvD (dihydroxyacid dehydratase) [4Fe-4S] cluster as an example. Combining a sensitive NO electrode with EPR (electron paramagnetic resonance) spectroscopy and an enzyme activity assay, we demonstrate that NO is rapidly consumed by the IlvD [4Fe-4S] cluster with the concomitant formation of the IlvD-bound DNIC (dinitrosyl-iron complex) and inactivation of the enzyme activity under anaerobic conditions. The rate constant for the initial reaction between NO and the IlvD [4Fe-4S] cluster is estimated to be (7.0+/-2.0)x10(6) M(-2) x s(-1) at 25 degrees C, which is approx. 2-3 times faster than that of the NO autoxidation by O2 in aqueous solution. Addition of GSH failed to prevent the NO-mediated modification of the IlvD [4Fe-4S] cluster regardless of the presence of O2 in the medium, further suggesting that NO is more reactive with the IlvD [4Fe-4S] cluster than with GSH or O2. Purified aconitase B [4Fe-4S] cluster from E. coli has an almost identical NO reactivity as the IlvD [4Fe-4S] cluster. However, the reaction between NO and the endonuclease III [4Fe-4S] cluster is relatively slow, apparently because the [4Fe-4S] cluster in endonuclease III is less accessible to solvent than those in IlvD and aconitase B. When E. coli cells containing recombinant IlvD, aconitase B or endonuclease III are exposed to NO using the Silastic tubing NO delivery system under aerobic and anaerobic conditions, the [4Fe-4S] clusters in IlvD and aconitase B, but not in endonuclease III, are efficiently modified forming the protein-bound DNICs, confirming that NO has a higher reactivity with the [4Fe-4S] clusters in IlvD and aconitase B than with O2 or GSH. The results suggest that the iron-sulfur clusters in proteins such as IlvD and aconitase B may constitute the primary targets of the NO cytotoxicity under both aerobic and anaerobic conditions.

Metabolic engineering of the L-valine biosynthesis pathway in Corynebacterium glutamicum using promoter activity modulation

The previously constructed strain Corynebacterium glutamicum ilvNM13 with acetohydroxy acid synthase, resistant to inhibition by all three branched-chain amino acids (L-valine, L-isoleucine and L-leucine), was used as a basis to develop a new type of valine producer by genetic engineering. The main strategy was to modulate expression of the genes involved in the biosynthesis of branched-chain amino acids. The activity of the promoters P- ilvD (dihydroxyacid dehydratase) and P- ilvE (transaminase) was up-modulated and the activity of the promoters P- ilvA (threonine deaminase) and P- leuA (isopropylmalate synthase) was down-modulated by site-directed mutagenesis. A constructed weak promoter of ilvA (or leuA), which was introduced into the C. glutamicum chromosome via a gene-replacement technique reduced the biosynthetic rate of isoleucine (or leucine), which lowered the mutant growth rate and increased valine production. Overexpression of ilvD and ilvE driven by the strong mutant promoters P- ilvDM7 and P- ilvEM6 resulted in an even higher level of valine production. Thus, the strain C. glutamicum ilvNM13 Δ panB P- ilvAM1CG P- ilvDM7 P- ilvEM6, having all mutations constructed within the chromosome, produced 136 mM valine in a 48-h cultivation.

Nitric oxide‐induced bacteriostasis and modification of iron‐sulphur proteins in Escherichia coli

The nitric oxide (NO) cytotoxicity has been well documented in bacteria and mammalian cells. However, the underlying mechanism is still not fully understood. Here we report that transient NO exposure effectively inhibits cell growth of Escherichia coli in minimal medium under anaerobic growth conditions and that cell growth is restored when the NO-exposed cells are either supplemented with the branched-chain amino acids (BCAA) anaerobically or returned to aerobic growth conditions. The enzyme activity measurements show that dihydroxyacid dehydratase (IlvD), an iron-sulphur enzyme essential for the BCAA biosynthesis, is completely inactivated in cells by NO with the concomitant formation of the IlvD-bound dinitrosyl iron complex (DNIC). Fractionation of the cell extracts prepared from the NO-exposed cells reveals that a large number of different protein-bound DNICs are formed by NO. While the IlvD-bound DNIC and other protein-bound DNICs are stable in cells under anaerobic growth conditions, they are efficiently repaired under aerobic growth conditions even without new protein synthesis. Additional studies indicate that L-cysteine may have an important role in repairing the NO-modified iron-sulphur proteins in aerobically growing E. coli cells. The results suggest that cellular deficiency to repair the NO-modified iron-sulphur proteins may directly contribute to the NO-induced bacteriostasis under anaerobic conditions.

Identification and characterization of the bacterial d-gluconate dehydratase in Achromobacter xylosoxidans

Achromobacter xylosoxidans is known to utilize d-glucose via the modified Entner-Doudoroff pathway. Although d-gluconate dehydratase produced from this bacterium was purified and partially characterized previously, a gene that encodes this enzyme has not yet been identified. To obtain protein information on bacterial d-gluconate dehydratase, we partially purified d-gluconate dehydratase in A. xylosoxidans and investigated its biochemical properties. Two degenerate primers were designed based on the N-terminal amino acid sequence of the partially purified d-gluconate dehydratase. Through PCR performed using degenerate primers, a 1,782-bp DNA sequence encoding the A. xylosoxidansd-gluconate dehydratase (gnaD) was obtained. The deduced amino acid sequence of A. xylosoxidans gnaD showed strong similarity with that of proteins belonging to the dihydroxy-acid dehydratase/phosphogluconate dehydratase family (COG0129). This is in contrast to the archaeal d-gluconate dehydratase, which belongs to the enolase superfamily (COG4948). The phylogenetic tree showed that A. xylosoxidansd-gluconate dehydratase is closer to the 6-phosphogluconate dehydratase than the dihydroxy-acid dehydratase. Interestingly, a clade containing A. xylosoxidans enzyme was clustered with proteins annotated as a second and a third dihydroxy-acid dehydratase in the genomes of Clostridium acetobutylicum (Cac_ilvD2) and Streptomyces ceolicolor (Sco_ilvD2, Sco_ilvD3), indicating that the function of these enzymes is the dehydration of d-gluconate.

Integrated network analysis identifies nitric oxide response networks and dihydroxyacid dehydratase as a crucial target in Escherichia coli

Nitric oxide (NO) is used by mammalian immune systems to counter microbial invasions and is produced by bacteria during denitrification. As a defense, microorganisms possess a complex network to cope with NO. Here we report a combined transcriptomic, chemical, and phenotypic approach to identify direct NO targets and construct the biochemical response network. In particular, network component analysis was used to identify transcription factors that are perturbed by NO. Such information was screened with potential NO reaction mechanisms and phenotypic data from genetic knockouts to identify active chemistry and direct NO targets in Escherichia coli. This approach identified the comprehensive E. coli NO response network and evinced that NO halts bacterial growth via inhibition of the branched-chain amino acid biosynthesis enzyme dihydroxyacid dehydratase. Because mammals do not synthesize branched-chain amino acids, inhibition of dihydroxyacid dehydratase may have served to foster the role of NO in the immune arsenal.

Crucial Role of Antioxidant Proteins and Hydrolytic Enzymes in Pathogenicity of Penicillium expansum: Analysis Based on Proteomics Approach

Penicillium expansum, a widespread filamentous fungus, is a major causative agent of fruit decay and may lead to the production of mycotoxin that causes harmful effects on human health. In this study, we compared the cellular and extracellular proteomes of P. expansum in the absence and presence of borate, which affects the virulence of the fungal pathogen. The differentially expressed proteins were identified using ESI-Q-TOF-MS/MS. Several proteins related to stress response (glutathione S-transferase, catalase, and heat shock protein 60) and basic metabolism (glyceraldehyde-3-phosphate dehydrogenase, dihydroxy-acid dehydratase, and arginase) were identified in the cellular proteome. Catalase and glutathione S-transferase, the two antioxidant enzymes, exhibited reduced levels of expression upon exposure to borate. Because catalase and glutathione S-transferase are related to oxidative stress response, we further investigated the reactive oxygen species (ROS) levels and oxidative protein carbonylation (damaged proteins) in P. expansum. Higher amounts of ROS and carbonylated proteins were observed after borate treatment, indicating that catalase and glutathione S-transferase are important in scavenging ROS and protecting cellular proteins from oxidative damage. Additionally to find secretory proteins that contribute to the virulence, we studied the extracellular proteome of P. expansum under stress condition with reduced virulence. The expression of three protein spots were repressed in the presence of borate and identified as the same hydrolytic enzyme, polygalacturonase.

Catalytic Promiscuity in Dihydroxy-Acid Dehydratase from the Thermoacidophilic Archaeon Sulfolobus solfataricus

Dihydroxy-acid dehydratase (DHAD) is one of the key enzymes involved in the biosynthetic pathway of the branched chain amino acids. Although the enzyme has been purified and characterized in various mesophiles, including bacteria and eukarya, the biochemical properties of DHAD from hyperthermophilic archaea have not yet been reported. In this study we cloned, expressed in Escherichia coli, and purified a DHAD homologue from the thermoacidophilic archaeon Sulfolobus solfataricus, which grows optimally at 80 degrees C and pH 3. The recombinant S. solfataricus DHAD (rSso_DHAD) showed the highest activity on 2,3-dihydroxyisovalerate among 17 aldonic acids tested. Interestingly, this enzyme also displayed high activity toward d-gluconate and some other pentonic and hexonic sugar acids. The k(cat)/K(m) values were 140.3 mM(-1) s(-1) for 2,3-dihydroxyisovalerate and 20.0 mM(-1) s(-1) for d-gluconate, respectively. A possible evolutionary explanation for substrate promiscuity was provided through amino acid sequence alignments of DHADs and 6-phosphogluconate dehydratases from archaea, bacteria and eukarya.

Characterisation of the enzyme activities involved in the valine biosynthetic pathway in a valine-producing strain of Corynebacterium glutamicum

The enzyme activities of the valine biosynthetic pathway and their regulation have been studied in the valine-producing strain, Corynebacterium glutamicum 13032Δ ilvApJC1 ilvBNCD. In this micro-organism, this pathway might involve up to five enzyme activities: acetohydroxy acid synthase (AHAS), acetohydroxy acid isomeroreductase (AHAIR), dihydroxyacid dehydratase and transaminases B and C. For each enzyme, kinetic parameters (optimal temperature, optimal pH and affinity for substrates) were determined. The first enzyme of the pathway, AHAS, was shown to exhibit a weak affinity for pyruvate (K m=8.3 mM). It appeared that valine and leucine inhibited the three first steps of the pathway (AHAS, AHAIR and DHAD). Moreover, the AHAS activity was inhibited by isoleucine. Considering the kinetic data collected during this work, AHAS would be a key enzyme for further strain improvement intending to increase the valine production by C. glutamicum.

Characterization of human cd200 glycoprotein receptor gene located on chromosome 3q12-13

An immunomodulatory membrane protein, CD200R displays an expression pattern restricted to myeloid cells in mice. It is the receptor for a ligand, CD200, expressed by a broad range of cell types. In this study, we describe the cloning and characterization of the human homologue of the CD200R gene. This gene maps closely to the CD200 gene on human chromosome 3q12-13. The human CD200R gene spans a region of 52 kb, consists of nine exons, and encodes a 348-amino-acid cell-surface protein consisting of two IgFF domains in a typical V/C2 arrangement. The 59-amino-acid cytoplasmic domain has two tyrosine residues, one of which is contained within a NPXY motif. In common with other IgSF genes, the CD200R gene can generate different protein isoforms through alternative splicing. An alternative spliceout form, which has not yet been described in mice, encodes a 188-amino-acid truncated soluble polypeptide containing only the V immunoglobulin domain. In contrast to murine CD200R protein, the human membrane-bound and soluble CD200R proteins have an insertion of 23 amino acids at position 23, encoded by exon 2, which generates a putative dihydroxyacid dehydratase domain. The splicing of exon 2 generates two new isoforms, encoding the membrane and soluble proteins but lacking the dyhydroxyacid dehydratase domain. Northern-blot analysis shows that both membrane-bound and soluble isoforms are expressed in the thymus, liver, spleen and placenta. By RT-PCR, we have analyzed the expression of the four transcript variants in human placenta, spleen, liver, brain and kidney.

Finding of an isoleucine derivative of a recombinant protein for pharmaceutical use

Protein modification generally occurs by addition to the amino acid side-chains of protein at the post-translational stage, for example, by enzymatic or chemical reactions after polypeptide synthesis. Recently, the recombinant hirudin analog CX-397, a potent thrombin inhibitor, was found to contain methylated Ile residues when it was overproduced by Escherichia coli in the absence of amino acids in the culture medium. The Ile derivatives, deduced to be β-methylnorleucine [βMeNle; (2 S, 3 S)-2-amino-3-methylhexanoic acid] by systematic chromatographic analysis, do not appear to be normal post-translational modifications of the protein because Ile has no functional group in its side-chain. We, therefore, propose that βMeNle is biosynthesized by E. coli, activated by E. coli isoleucyl-tRNA synthetase (IleRS), then incorporated into the overproduced recombinant hirudin analog. The biosynthesis of βMeNle in E. coli is thought to occur as follows: α-ketovalerate is synthesized from α-ketobutyrate by three Leu biosynthetic enzymes, α-isopropylmalate synthase (IPMS) (EC 4.1.3.12), α-isopropylmalate isomerase (ISOM) (EC 4.2.1.33) and β-isopropylmalate dehydrogenase (IPMD) (EC 1.1.1.85), which have broad substrate specificities. α-Ketovalerate is then converted to α-keto-β-methylcaproate by three Ile and Val biosynthetic enzymes, acetohydroxy acid synthase (AS) (EC 4.1.3.18), acetohydroxy acid isomeroreductase (IR) (EC 1.1.1.86) and dihydroxy acid dehydratase (DH) (EC 4.2.1.9). Finally, this is converted to βMeNle by branched-chain amino acid transaminase (EC 2.6.1.42), one of the Ile and Val biosynthetic enzymes.

Oxygen toxicity from plants to people.

Over the past 30 years, acute oxygen toxicity in plants, mammals and enteric bacteria has been defined in terms of specific interactions of oxygen with a limited number of molecular targets. At least in the case of plants and mammals, response at the level of the whole organism is a consequence of oxygen's interaction with enzymes that should not exhibit oxygen sensitivity, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) and glutamate decarboxylase (GAD). In enteric bacteria, inhibition of acetolactate synthase (ALS), or the production of peracetic acid by this enzyme, may be a contributing factor in the inactivation of dihydroxyacid dehydratase and loss of the ability to synthesize branched-chain amino acids under conditions of hyperbaric oxygen. The facile interaction of these enzymes with oxygen has questioned our fundamental understanding of their reaction mechanisms. Could these enzymes have radical mechanisms?