Branched-chain Amino Acid Biosynthesis Pathway Research Articles

2-Hydroxyisovalerate production by Klebsiella pneumoniae

2-Hydroxyisovalerate is a valuable chemical that can be used in the production of biodegradable polyesters. In nature, it was only produced at a very low level by Lactococcus lactis. 2-Ketoisovalerate is an intermediate metabolite of the branched-chain amino acid biosynthesis pathway, and Klebsiella pneumoniae ΔbudAΔldhA (Kp ΔbudAΔldhA) was a 2-ketoisovalerate producing strain. In this research, 2-hydroxyisovalerate was identified as a metabolite of Kp ΔbudAΔldhA, and its synthesis pathway was revealed. It was found that 2-ketoisovalerate and 2-hydroxyisovalerate were produced by Kp ΔbudA and Kp ΔbudAΔldhA, but not by Kp ΔbudAΔldhAΔilvD in which the 2-ketoisovalerate synthesis was blocked. budA, ldhA, and ilvD encode α-acetolactate decarboxylase, lactate dehydrogenase, and dihydroxy acid dehydratase, respectively. Thus, it was deduced that 2-hydroxyisovalerate was synthesized from 2-ketoisovalerate. Isoenzymes of ketopantoate reductase PanE, PanE2, and IlvC were suspected of being responsible for this reaction. Kinetic parameters of these enzymes were detected, and they all hold the 2-ketoisovalerate reductase activities. PanE and PanE2 use both NADH and NADPH as co-factors. While IlvC only uses NADH as a co-factor. Over-expression of panE, panE2, or ilvC in Kp ΔbudAΔldhA all enhanced the production of 2-hydroxyisovalerate. Accordingly, 2-hydroxyisovalerate levels were reduced by knocking out panE or panE2. In fed-batch fermentation, 14.41 g/L of 2-hydroxyisovalerate was produced by Kp ΔbudAΔldhA-panE, with a substrate conversion ratio of 0.13 g/g glucose.

Whole-transcriptome RNA sequencing reveals changes in amino acid metabolism induced in harvested broccoli by red LED irradiation

Whole-transcriptomic profiling combined with amino acid analysis were conducted in order to gain a better understanding of global changes in amino acid metabolism induced in broccoli by red LED irradiation. The results showed that the contents of almost all 16 amino acids in postharvest broccoli were maintained under red LED illumination. The red LED irradiation enhanced the anabolism of amino acid, including the biosynthesis of aromatic amino acids by upregulating the genes’ expression in the shikimate pathway, as well as by upregulating the genes’ expression which encoding biosynthetic enzymes in the branched-chain amino acid biosynthesis pathway. Red LED irradiation induced the expression of genes encoding aspartate aminotransferase, which plays a role in Asp synthesis, aspartate kinase, which functions in aspartate metabolism, and a cytoplasmic aspartate aminotransferase that converts 2-Oxoglutarate into Glu. Genes encoding imidazole glycerol-phosphate synthase and histidinol-phosphatase, which function in the His biosynthesis pathway, were also upregulated. According to our results, red LED irradiation delays broccoli’s yellowing and senescence by regulating amino acid metabolism. These results enhance our understanding of the role of amino acid metabolism in the senescence of broccoli and the mechanism of red LED irradiation to alter amino acid metabolism in harvested broccoli.

Dihydroxy-Acid Dehydratases From Pathogenic Bacteria: Emerging Drug Targets to Combat Antibiotic Resistance.

There is an urgent global need for the development of novel therapeutics to combat the rise of various antibiotic‐resistant superbugs. Enzymes of the branched‐chain amino acid (BCAA) biosynthesis pathway are an attractive target for novel anti‐microbial drug development. Dihydroxy‐acid dehydratase (DHAD) is the third enzyme in the BCAA biosynthesis pathway. It relies on an Fe−S cluster for catalytic activity and has recently also gained attention as a catalyst in cell‐free enzyme cascades. Two types of Fe−S clusters have been identified in DHADs, i.e. [2Fe−2S] and [4Fe−4S], with the latter being more prone to degradation in the presence of oxygen. Here, we characterise two DHADs from bacterial human pathogens, Staphylococcus aureus and Campylobacter jejuni (SaDHAD and CjDHAD). Purified SaDHAD and CjDHAD are virtually inactive, but activity could be reversibly reconstituted in vitro (up to ∼19,000‐fold increase with kcat as high as ∼6.7 s−1). Inductively‐coupled plasma‐optical emission spectroscopy (ICP‐OES) measurements are consistent with the presence of [4Fe−4S] clusters in both enzymes. N‐isopropyloxalyl hydroxamate (IpOHA) and aspterric acid are both potent inhibitors for both SaDHAD (K i=7.8 and 51.6 μM, respectively) and CjDHAD (K i=32.9 and 35.1 μM, respectively). These compounds thus present suitable starting points for the development of novel anti‐microbial chemotherapeutics.

Abstract 3617: METTL16 drives leukemogenesis and maintains leukemia stem cell self-renewal via reprogramming BCAA metabolism

Abstract Background: As the most prevalent internal decorations in mammalian mRNA, N6-methyladenosine (m6A) has been reported to be involved in many physiological and pathological processes, including acute myeloid leukemia (AML). METTL3 and METTL14, the well-recognized m6A methyltransferase complex, contribute to AML. METTL16 is a recently identified m6A methyltransferase that has been reported to deposit m6A in a few targets. While, unlike METTL3/14, the biological functions of METTL16 are largely unknown. Here, we explored the function and mechanism of METTL16 in AML pathogenesis and evaluated its therapeutic potential for AML treatment. Methods: We performed CRISPR-Cas9 screen to evaluate the dependency of METTL16 in AML cells. We created METTL16 knockout (KO) cells and conditional KO mice to evaluate its role in leukemogenesis and normal hematopoiesis. We employed bone marrow transplantation (BMT), xenograft, and AML patient-derived xenograft (PDX) models to determine its role in AML development and progression. To identify the targets of METTL16, we performed m6A-seq and RNA-seq, followed m6A-qPCR, CLIP-qPCR, in vitro methyltransferase assays and RNA stability assays. To examine the effect of METTL16 on branched chain amino acid (BCAA) metabolism, we performed metabolic profiling with 13C, 15N-leucine. Results: CRISPR-Cas9 screen showed METTL16 is one of the most essential genes for the survival of AMLs. The AML cells display more robust dependency on METTL16 than METTL3/14. We found METTL16 is highly expressed in AML patients compared to healthy controls. METTL16 KO significantly inhibited AML cell proliferation, promoted cell apoptosis and myeloid differentiation in vitro, which could be totally reversed by forced expression of wild-type METTL16, but not catalytic-dead mutant. METTL16 depletion dramatically inhibited AML progression and prolonged survival of recipient mice in the BMT, xenograft and PDX models. In addition, METTL16 is highly expressed in LSCs contrast to leukemic bulk cells and METTL16 KO significantly attenuates LSC self-renewal in vitro and in vivo. By contrast, the role of METLL16 is largely spared in normal hematopoietic cells. Via integrated analysis of m6A-seq data and RNA-seq data, we identified two bona fide targets of METTL16, BCAT1 and BCAT2, which encode two critical BCAA transaminases in BCAA biosynthesis pathway. METTL16 promotes the expression of BCAT1 and BCAT2 via an m6A dependent manner. Metabolomics with 13C, 15N-leucine tracing showed that METTL16 KO results in suppressed pools of TCA cycle intermediates, some non-essential amino acids and nucleotides. Conclusion: We uncover a tumor-promoting role of METTL16 in AML and LSC self-renewal via reprogramming BCAA metabolism, in which METTL16 functions as an m6A methyltransferase to regulate expression of BCAT1 and BCAT2. Our data suggest that METTL16 is an attractive target for AML therapy. Citation Format: Li Han, Lei Dong, Keith Leung, Zhicong Zhao, Yangchan Li, Ying Qing, Jianhuang Xue, Chao Shen, Zhenhua Chen, Lei Gao, Kitty Wang, Keren Zhou, Wei Li, Brandon Tan, Zheng Zhang, Xi Qin, Rui Su, Xiaolan Deng, Jianjun Chen. METTL16 drives leukemogenesis and maintains leukemia stem cell self-renewal via reprogramming BCAA metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3617.

Discovery, Synthesis and Evaluation of a Ketol-Acid Reductoisomerase Inhibitor.

Ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid biosynthesis pathway, is a potential drug target for bacterial infections including Mycobacterium tuberculosis. Here, we have screened the Medicines for Malaria Venture Pathogen Box against purified M. tuberculosis (Mt) KARI and identified two compounds that have Ki values below 200 nm. In Mt cell susceptibility assays one of these compounds exhibited an IC50 value of 0.8 μm. Co-crystallization of this compound, 3-((methylsulfonyl)methyl)-2H-benzo[b][1,4]oxazin-2-one (MMV553002), in complex with Staphylococcus aureus KARI, which has 56 % identity with Mt KARI, NADPH and Mg2+ yielded a structure to 1.72 Å resolution. However, only a hydrolyzed product of the inhibitor (i.e. 3-(methylsulfonyl)-2-oxopropanic acid, missing the 2-aminophenol attachment) is observed in the active site. Surprisingly, Mt cell susceptibility assays showed that the 2-aminophenol product is largely responsible for the anti-TB activity of the parent compound. Thus, 3-(methylsulfonyl)-2-oxopropanic acid was identified as a potent KARI inhibitor that could be further explored as a potential biocidal agent and we have shown 2-aminophenol, as an anti-TB drug lead, especially given it has low toxicity against human cells. The study highlights that careful analysis of broad screening assays is required to correctly interpret cell-based activity data.

Differential Impacts of Gingerols- and Shogaols-Enriched Ginger Root Extracts on Fecal Metabolites in Rats with Neuropathic Pain

Abstract Objectives Emerging evidence suggests that gut microbiota-derived mediators play key roles in the development of chronic pain, particularly, neuropathic pain (NP), by serving as critical modulators. We evaluated the effects of two ginger root extracts, gingerols and shogaols respectively, on gut microbiota-derived metabolites in the feces of animals with NP. Methods Sixteen male rats were randomly divided into 4 groups: sham group, SNL group, SNL+gingerols-enriched ginger (GEG) extract group, and SNL+shogoals-enriched ginger (SEG) extract group. At 30 days, fecal samples were processed or LC-MS/MS analysis. Principle Component Analysis was performed to assess the different profiling of the metabolites detected and quantified among 4 groups. Data were analyzed using compound discover software (3.1) to identify and quantify metabolites. t-test was performed to determine the number of statistically different metabolites in SNL group compared to the other groups. Results Among all identified pathways, three amino acid- associated pathways (namely, branched chain amino acid biosynthesis (BCAAB), anaerobic aromatic compound degradation (AACD), and aromatic amino acid biosynthesis (AAAB)) were significantly affected in the paired comparisons. The SNL group tended to have higher concentration of phenylacetadehyde (AACD pathway) than that in the sham group (P = 0.09). In BCAAB pathway, relative to the SNL group, (i) GEG group had an increase in L-glutamate, 4-methyl-2-oxopentanoate, and (S)-3-methyl-2-oxopentanoate, and a decrease in (S)-2-acetolactate, and (ii) SEG group showed increased L-valine and decreased 2-oxoglutarate levels. In AACD pathway, compared to the SNL group, R-2-benzylsuccinate, phenylacetate, phenlacetadehyde, and L-glutamate levels were increased in the GEG group, while only phenlacetate level was increased in the SEG group. In AAAB pathway, compared to the SNL group, increased L-glutamate and decreased D-erythrose 4-phospahte level was seen in the GEG group; whereas the SEG group tended to have lower fecal L-glutamine level. Conclusions Based on the results of fecal metabolite analyses, gingerols and shogaols supplementation had significant but differential impacts on the amino acid-associated pathways. Funding Sources Texas Tech University Health Sciences Center.

The ilvGMEDA Operon Is Regulated by Transcription Attenuation in Vibrio alginolyticus ZJ-T.

Bacteria synthesize amino acids according to their availability in the environment or, in the case of pathogens, within the host. We explored the regulation of the biosynthesis of branched-chain amino acids (BCAAs) (l-leucine, l-valine, and l-isoleucine) in Vibrio alginolyticus, a marine fish and shellfish pathogen and an emerging opportunistic human pathogen. In this species, the ilvGMEDA operon encodes the main pathway for biosynthesis of BCAAs. Its upstream regulatory region shows no sequence similarity to the corresponding region in Escherichia coli or other Enterobacteriaceae, and yet we show that this operon is regulated by transcription attenuation. The translation of a BCAA-rich peptide encoded upstream of the structural genes provides an adaptive response similar to the E. coli canonical model. This study of a nonmodel Gram-negative organism highlights the mechanistic conservation of transcription attenuation despite the absence of primary sequence conservation.IMPORTANCE This study analyzes the regulation of the biosynthesis of branched-chain amino acids (leucine, valine, and isoleucine) in Vibrio alginolyticus, a marine bacterium that is pathogenic to fish and humans. The results highlight the conservation of the main regulatory mechanism with that of the enterobacterium Escherichia coli, suggesting that such a mechanism appeared early during the evolution of Gram-negative bacteria, allowing adaptation to a wide range of environments.

Discovery and evaluation of novel Mycobacterium tuberculosis ketol-acid reductoisomerase inhibitors as therapeutic drug leads.

Tuberculosis (TB) remains a major threat to human health. This due to the fact that current drug treatments are less than optimal and the increasing occurrence of multi drug-resistant strains of etiological agent, Mycobacterium tuberculosis (Mt). Given the wide-spread significance of this disease, we have undertaken a design and evaluation program to discover new anti-TB drug leads. Here, we focused on ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid biosynthesis pathway. Importantly, this enzyme is present in bacteria but not in humans, making it an attractive proposition for drug discovery. In the present work, we used molecular docking to identify seventeen potential inhibitors of KARI using an in-house database. Compounds were selected based on docking scores, which were assigned as the result of favourable interactions between the compound and the active site of KARI. The inhibitory constant values for two leads, compounds 14 and 16 are 3.71 and 3.06µM respectively. To assess the mode of binding, 100ns molecular dynamics simulations for these two compounds in association with Mt KARI were performed and showed that the complex was stable with an average root mean square deviation of less than 3.5Å for all atoms. Furthermore, compound 16 showed a minimum inhibitory concentration of 2.06 ± 0.91µM and a 1.9 fold logarithmic reduction in the growth of Mt in an infected macrophage model. The two compounds exhibited low toxicity against RAW 264.7 cell lines. Thus, both compounds are promising candidates for development as an anti-TB drug leads.

Induction of the PDH bypass and upregulation of the ALDH7B4 in plants treated with herbicides inhibiting amino acid biosynthesis

Imazamox and glyphosate represent two classes of herbicides that inhibit the activity of acetohydroxyacid synthase in the branched-chain amino acid biosynthesis pathway and the activity of 5-enolpyruvylshikimate-3-phosphate synthase in the aromatic amino acid biosynthesis pathway, respectively. However, it is still unclear how imazamox and glyphosate lead to plant death. Both herbicides inhibit amino-acid biosynthesis and were found to induce ethanol fermentation in plants, but an Arabidopsis mutant deficient in alcohol dehydrogenase 1 was neither more susceptible nor more resistant than the wild-type to the herbicides. In this study, we investigated the effects of the amino acid biosynthesis inhibitors, imazamox and glyphosate, on the pyruvate dehydrogenase bypass reaction and fatty acid metabolism in A. thaliana. We found that the pyruvate dehydrogenase bypass was upregulated following the treatment by the two herbicides. Our results suggest that the Arabidopsis aldehyde dehydrogenase 7B4 gene might be participating in the pyruvate dehydrogenase bypass reaction. We evaluated the potential role of the aldehyde dehydrogenase 7B4 upon herbicide treatment in the plant defence mechanism. Plants that overexpressed the ALDH7B4 gene accumulated less soluble sugars, starch, and fatty acids and grew better than the wild-type after herbicide treatment. We discuss how the upregulation of the ALDH7B4 alleviates the effects of the herbicides, potentially through the detoxification of the metabolites produced in the pyruvate dehydrogenase bypass.

Crystal structure of plant acetohydroxyacid synthase, the target for several commercial herbicides.

The coordinates and structure factors for uninhibited AtAHAS have been deposited in the Protein Data Bank (www.pdb.org) with the PDB ID code 5K6Q.

Comprehensive understanding of acetohydroxyacid synthase inhibition by different herbicide families

Five commercial herbicide families inhibit acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6), which is the first enzyme in the branched-chain amino acid biosynthesis pathway. The popularity of these herbicides is due to their low application rates, high crop vs. weed selectivity, and low toxicity in animals. Here, we have determined the crystal structures of Arabidopsis thaliana AHAS in complex with two members of the pyrimidinyl-benzoate (PYB) and two members of the sulfonylamino-carbonyl-triazolinone (SCT) herbicide families, revealing the structural basis for their inhibitory activity. Bispyribac, a member of the PYBs, possesses three aromatic rings and these adopt a twisted "S"-shaped conformation when bound to A. thaliana AHAS (AtAHAS) with the pyrimidinyl group inserted deepest into the herbicide binding site. The SCTs bind such that the triazolinone ring is inserted deepest into the herbicide binding site. Both compound classes fill the channel that leads to the active site, thus preventing substrate binding. The crystal structures and mass spectrometry also show that when these herbicides bind, thiamine diphosphate (ThDP) is modified. When the PYBs bind, the thiazolium ring is cleaved, but when the SCTs bind, ThDP is modified to thiamine 2-thiazolone diphosphate. Kinetic studies show that these compounds not only trigger reversible accumulative inhibition of AHAS, but also can induce inhibition linked with ThDP degradation. Here, we describe the features that contribute to the extraordinarily powerful herbicidal activity exhibited by four classes of AHAS inhibitors.

Metal Ions Play an Essential Catalytic Role in the Mechanism of Ketol-Acid Reductoisomerase.

Ketol-acid reductoisomerase (KARI) is a Mg(2+) -dependent enzyme in the branched-chain amino acid biosynthesis pathway. It catalyses a complex two-part reaction: an alkyl migration followed by a NADPH-dependent reduction. Both reactions occur within the one active site, but in particular, the mechanism of the isomerisation step is poorly understood. Here, using a combination of kinetic, thermodynamic and spectroscopic techniques, the reaction mechanisms of both Escherichia coli and rice KARI have been investigated. We propose a conserved mechanism of catalysis, whereby a hydroxide, bridging the two Mg(2+) ions in the active site, initiates the reaction by abstracting a proton from the C2 alcohol group of the substrate. While the μ-hydroxide-bridged dimetallic centre is pre-assembled in the bacterial enzyme, in plant KARI substrate binding leads to a reduction of the metal-metal distance with the concomitant formation of a hydroxide bridge. Only Mg(2+) is capable of promoting the isomerisation reaction, likely to be due to non-competent substrate binding in the presence of other metal ions.

New insight into the photoheterotrophic growth of the isocytrate lyase-lacking purple bacterium Rhodospirillum rubrum on acetate.

Purple non-sulfur bacteria are well known for their metabolic versatility. One of these bacteria, Rhodospirillum rubrum S1H, has been selected by the European Space Agency to ensure the photoheterotrophic assimilation of volatile fatty acids in its regenerative life support system, MELiSSA. Here, we combined proteomic analysis with bacterial growth analysis and enzymatic activity assays in order to better understand acetate photoassimilation. In this isocitrate lyase-lacking organism, the assimilation of two-carbon compounds cannot occur through the glyoxylate shunt, and the citramalate cycle has been proposed to fill this role, while, in Rhodobacter sphaeroides, the ethylmalonyl-CoA pathway is used for acetate assimilation. Using proteomic analysis, we were able to identify and quantify more than 1700 unique proteins, representing almost one-half of the theoretical proteome of the strain. Our data reveal that a pyruvate : ferredoxin oxidoreductase (NifJ) could be used for the direct assimilation of acetyl-CoA through pyruvate, potentially representing a new redox-balancing reaction. We additionally propose that the ethylmalonyl-CoA pathway could also be involved in acetate assimilation by the examined strain, since specific enzymes of this pathway were all upregulated and activity of crotonyl-CoA reductase/carboxylase was increased in acetate conditions. Surprisingly, we also observed marked upregulation of glutaryl-CoA dehydrogenase, which could be a component of a new pathway for acetate photoassimilation. Finally, our data suggest that citramalate could be an intermediate of the branched-chain amino acid biosynthesis pathway, which is activated during acetate assimilation, rather than a metabolite of the so-called citramalate cycle.

Sulfonylureas Have Antifungal Activity and Are Potent Inhibitors of Candida albicans Acetohydroxyacid Synthase

The sulfonylurea herbicides exert their activity by inhibiting plant acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway. It has previously been shown that if the gene for AHAS is deleted in Candida albicans , attenuation of virulence is achieved, suggesting AHAS as an antifungal drug target. Herein, we have cloned, expressed, and purified C. albicans AHAS and shown that several sulfonylureas are inhibitors of this enzyme and possess antifungal activity. The most potent of these compounds is ethyl 2-(N-((4-iodo-6-methoxypyrimidin-2-yl)carbamoyl)sulfamoyl)benzoate (10c), which has a K(i) value of 3.8 nM for C. albicans AHAS and an MIC₉₀ of 0.7 μg/mL for this fungus in cell-based assays. For the sulfonylureas tested there was a strong correlation between inhibitory activity toward C. albicans AHAS and fungicidal activity, supporting the hypothesis that AHAS is the target for their inhibitory activity within the cell.

Bacterial and Plant Ketol-Acid Reductoisomerases Have Different Mechanisms of Induced Fit during the Catalytic Cycle

Ketol-acid reductoisomerase (KARI) is the second enzyme in the branched-chain amino acid biosynthesis pathway, which is found in plants, fungi and bacteria but not in animals. This difference in metabolism between animals and microorganisms makes KARI an attractive target for the development of antimicrobial agents. Herein we report the crystal structure of Escherichia coli KARI in complex with Mg(2+) and NADPH at 2.3Å resolution. Ultracentrifugation studies confirm that the enzyme exists as a tetramer in solution, and isothermal titration calorimetry shows that the binding of Mg(2+) increases structural disorder while the binding of NADPH increases the structural rigidity of the enzyme. Comparison of the structure of the E. coli KARI-Mg(2+)-NADPH complex with that of enzyme in the absence of cofactors shows that the binding of Mg(2+) and NADPH opens the interface between the N- and C-domains, thereby allowing access for the substrates to bind: the existence of only a small opening between the domains in the crystal structure of the unliganded enzyme signifies restricted access to the active site. This observation contrasts with that in the plant enzyme, where the N-domain can rotate freely with respect to the C-domain until the binding of Mg(2+) and/or NADPH stabilizes the relative positions of these domains. Support is thereby provided for the idea that plant and bacterial KARIs have evolved different mechanisms of induced fit to prepare the active site for catalysis.

Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha

Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180 mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the production titer improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14 g/L branched-chain alcohols over the duration of 50 days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcohols and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.

Preliminary X-ray crystallographic studies of the catalytic subunit ofEscherichia coliAHAS II with its cofactors

Acetohydroxyacid synthase (AHAS) is the first common enzyme in the branched-chain amino-acid biosynthesis pathway and is the target of several classes of commercial herbicides. In this study, the Escherichia coli ilvG gene that encodes the catalytic subunit of AHAS II was cloned into the pET28a vector and expressed in soluble form at high levels in E. coli strain BL21 (DE3) cells. The protein was purified using Ni(2+)-chelating chromatography followed by size-exclusion chromatography. The catalytic subunit of E. coli AHAS II was cocrystallized with its cofactors Mg(2+), FAD and ThDP using the sitting-drop vapour-diffusion method and the crystals diffracted to 2.80 Å resolution.

Inactivation of the ilvB1 gene in Mycobacterium tuberculosis leads to branched-chain amino acid auxotrophy and attenuation of virulence in mice

Acetohydroxyacid synthase (AHAS) is the first enzyme in the branched-chain amino acid biosynthesis pathway in bacteria. Bioinformatics analysis revealed that the Mycobacterium tuberculosis genome contains four genes (ilvB1, ilvB2, ilvG and ilvX) coding for the large catalytic subunit of AHAS, whereas only one gene (ilvN or ilvH) coding for the smaller regulatory subunit of this enzyme was found. In order to understand the physiological role of AHAS in survival of the organism in vitro and in vivo, we inactivated the ilvB1 gene of M. tuberculosis. The mutant strain was found to be auxotrophic for all of the three branched-chain amino acids (isoleucine, leucine and valine), when grown with either C(6) or C(2) carbon sources, suggesting that the ilvB1 gene product is the major AHAS in M. tuberculosis. Depletion of these branched chain amino acids in the medium led to loss of viability of the DeltailvB1 strain in vitro, resulting in a 4-log reduction in colony-forming units after 10 days. Survival kinetics of the mutant strain cultured in macrophages maintained with sub-optimal concentrations of the branched-chain amino acids did not show any loss of viability, indicating either that the intracellular environment was rich in these amino acids or that the other AHAS catalytic subunits were functional under these conditions. Furthermore, the growth kinetics of the DeltailvB1 strain in mice indicated that although this mutant strain showed defective growth in vivo, it could persist in the infected mice for a long time, and therefore could be a potential vaccine candidate.

Conformational Changes in a Plant Ketol-Acid Reductoisomerase upon Mg2+ and NADPH Binding as Revealed by Two Crystal Structures

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) is an enzyme in the branched-chain amino acid biosynthesis pathway where it catalyzes the conversion of 2-acetolactate into (2R)-2,3-dihydroxy-3-isovalerate or the conversion of 2-aceto-2-hydroxybutyrate into (2R,3R)-2,3-dihydroxy-3-methylvalerate. KARI catalyzes two reactions—alkyl migration and reduction—and requires Mg2+ and NADPH for activity. To date, the only reported structures for a plant KARI are those of the spinach enzyme–Mn2+–(phospho)ADP ribose–(2R,3R)-2,3-dihydroxy-3-methylvalerate complex and the spinach KARI–Mg2+–NADPH–N-hydroxy-N-isopropyloxamate complex, where N-hydroxy-N-isopropyloxamate is a predicted transition-state analog. These studies demonstrated that the enzyme consists of two domains, N-domain and C-domain, with the active site at the interface of these domains. Here, we have determined the structures of the rice KARI–Mg2+ and rice KARI–Mg2+–NADPH complexes to 1.55 Å and 2.80 Å resolutions, respectively. In comparing the structures of all the complexes, several differences are observed. Firstly, the N-domain is rotated up to 15° relative to the C-domain, expanding the active site by up to 4 Å. Secondly, an α-helix in the C-domain that includes residues V510-T519 and forms part of the active site moves by ∼3.9 Å upon binding of NADPH. Thirdly, the 15 C-terminal amino acid residues in the rice KARI–Mg2+ complex are disordered. In the rice KARI–Mg2+–NADPH complex and the spinach KARI structures, many of the 15 residues bind to NADPH and the N-domain and cover the active site. Fourthly, the location of the metal ions within the active site can vary by up to 2.7 Å. The new structures allow us to propose that an induced-fit mechanism operates to (i) allow substrate to enter the active site, (ii) close over the active site during catalysis, and (iii) open the active site to facilitate product release.

Auxotrophy Accounts for Nodulation Defect of Most Sinorhizobium meliloti Mutants in the Branched-Chain Amino Acid Biosynthesis Pathway

Some Sinorhizobium meliloti mutants in genes involved in isoleucine, valine, and leucine biosynthesis were previously described as being unable to induce nodule formation on host plants. Here, we present a reappraisal of the interconnection between the branched-chain amino acid biosynthesis pathway and the nodulation process in S. meliloti. We characterized the symbiotic phenotype of seven mutants that are auxotrophic for isoleucine, valine, or leucine in two closely related S. meliloti strains, 1021 and 2011. We showed that all mutants were similarly impaired for nodulation and infection of the Medicago sativa host plant. In most cases, the nodulation phenotype was fully restored by the addition of the missing amino acids to the plant growth medium. This strongly suggests that auxotrophy is the cause of the nodulation defect of these mutants. However, we confirmed previous findings that ilvC and ilvD2 mutants in the S. meliloti 1021 genetic background could not be restored to nodulation by supplementation with exogenous amino acids even though their Nod factor production appeared to be normal.