Improvement of l-Leucine Production in Corynebacterium glutamicum by Altering the Redox Flux.

The production of l-leucine was improved by the disruption of ltbR encoding transcriptional regulator and overexpression of the key genes (leuAilvBNCE) of the l-leucine biosynthesis pathway in Corynebacterium glutamicum XQ-9. In order to improve l-leucine production, we rationally engineered C. glutamicum to enhance l-leucine production, by improving the redox flux. On the basis of this, we manipulated the redox state of the cells by mutating the coenzyme-binding domains of acetohydroxyacid isomeroreductase encoded by ilvC, inserting NAD-specific leucine dehydrogenase, encoded by leuDH from Lysinibacillus sphaericus, and glutamate dehydrogenase encoded by rocG from Bacillus subtilis, instead of endogenous branched-chain amino acid transaminase and glutamate dehydrogenase, respectively. The yield of l-leucine reached 22.62 ± 0.17 g·L-1 by strain ΔLtbR-acetohydroxyacid isomeroreductase (AHAIR)M/ABNCME, and the concentrations of the by-products (l-valine and l-alanine) increased, compared to the strain ΔLtbR/ABNCE. Strain ΔLtbR-AHAIRMLeuDH/ABNCMLDH accumulated 22.87±0.31 g·L-1 l-leucine, but showed a drastically low l-valine accumulation (from 8.06 ± 0.35 g·L-1 to 2.72 ± 0.11 g·L-1), in comparison to strain ΔLtbR-AHAIRM/ABNCME, which indicated that LeuDH has much specificity for l-leucine synthesis but not for l-valine synthesis. Subsequently, the resultant strain ΔLtbR-AHAIRMLeuDHRocG/ABNCMLDH accumulated 23.31 ± 0.24 g·L-1 l-leucine with a glucose conversion efficiency of 0.191 g·g-1.

A proteomic analysis of salt stress response in seedlings of two African rice cultivars.

Salt stress is one of the key abiotic stresses threatening future agricultural production and natural ecosystems. This study investigates the salt stress response of two rice seedlings, which were screened from 28 Kenya rice cultivars. A proteomic analysis was carried out and Mapman bin codes employed in protein function categorization. Proteins in the redox, stress, and signaling categories were identified, and whose expression differed between the salt tolerant and the salt sensitive samples employed in the present study. 104 and 102 root proteins were observed as significantly altered during salt stress in the tolerant and sensitive samples, respectively and 13 proteins were commonly expressed. Among the 13 proteins, ketol-acid reductoisomerase protein was upregulated in both 1 and 3days of salt treatment in the tolerant sample, while it was down-regulated in both 1 and 3days of salt treatment in the sensitive sample. Actin-7, tubulin alpha, V-type proton ATPase, SOD (Cu-Zn), SOD (Mn), and pyruvate decarboxylase were among the observed salt-induced proteins. In general, this study improves our understanding about salt stress response mechanisms in rice.

Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases.

Although most sequenced members of the industrially important ketol-acid reductoisomerase (KARI) family are class I enzymes, structural studies to date have focused primarily on the class II KARIs, which arose through domain duplication. In the present study, we present five new crystal structures of class I KARIs. These include the first structure of a KARI with a six-residue ß2αB (cofactor specificity determining) loop and an NADPH phosphate-binding geometry distinct from that of the seven- and 12-residue loops. We also present the first structures of naturally occurring KARIs that utilize NADH as cofactor. These results show insertions in the specificity loops that confounded previous attempts to classify them according to loop length. Lastly, we explore the conformational changes that occur in class I KARIs upon binding of cofactor and metal ions. The class I KARI structures indicate that the active sites close upon binding NAD(P)H, similar to what is observed in the class II KARIs of rice and spinach and different from the opening of the active site observed in the class II KARI of Escherichia coli. This conformational change involves a decrease in the bending of the helix that runs between the domains and a rearrangement of the nicotinamide-binding site.

General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH.

To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.

Effective trapping of fruit flies with cultures of metabolically modified acetic acid bacteria.

Acetoin in vinegar is an attractant to fruit flies when combined with acetic acid. To make vinegar more effective in attracting fruit flies with increased acetoin production, Komagataeibacter europaeus KGMA0119 was modified by specific gene disruption of the acetohydroxyacid isomeroreductase gene (ilvC). A previously constructed mutant lacking the putative ligand-sensing region in the leucine-responsive regulatory protein (KeLrp, encoded by Kelrp) was also used. The ilvC and Kelrp disruptants (KGMA5511 and KGMA7203, respectively) produced greater amounts of acetoin (KGMA5511, 0.11%; KGMA7203, 0.13%) than the wild-type strain KGMA0119 (0.069%). KGMA7203 produced a trace amount of isobutyric acid (0.007%), but the other strains did not. These strains produced approximately equal amounts of acetic acid (0.7%). The efficiency of fruit fly attraction was investigated with cultured Drosophila melanogaster. D. melanogaster flies (approximately 1,500) were released inside a cage (2.5 m by 2.5 m by 1.5 m) and were trapped with a device containing vinegar and a sticky sheet. The flies trapped on the sticky sheet were counted. The cell-free supernatant from KGMA7203 culture captured significantly more flies (19.36 to 36.96% of released flies) than did KGMA0119 (3.25 to 11.40%) and KGMA5511 (6.87 to 21.50%) cultures. Contrastingly, a 0.7% acetic acid solution containing acetoin (0.13%) and isobutyric acid (0.007%), which mimicked the KGMA7203 supernatant, captured significantly fewer flies (0.88 to 4.57%). Furthermore, the KGMA0119 supernatant with additional acetoin (0.13%) and isobutyric acid (0.007%) captured slightly more flies than the original KGMA0119 supernatant but fewer than the KGMA7203 supernatant, suggesting that the synergistic effects of acetic acid, acetoin, isobutyric acid, and unidentified metabolites achieved the efficient fly trapping of the KGMA7203 supernatant.

Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16.

2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the three R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by L-valine (IC50=1.2 mM), L-isoleucine (IC50=2.3 mM), and L-leucine (IC50=5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (KM=10.5 µM) and is highly selective towards 2-ketobutyrate (R=140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2-ketoisovalerate for value-added materials.

FgIlv5 is required for branched-chain amino acid biosynthesis and full virulence in Fusarium graminearum.

In this study, we characterized FgIlv5, a homologue of the Saccharomyces cerevisiae keto-acid reductoisomerase (KARI) from the important wheat head scab fungus Fusarium graminearum. KARI is a key enzyme in the branched-chain amino acid (BCAA, including leucine, isoleucine and valine) biosynthetic pathway that exists in a variety of organisms from bacteria to fungi and higher plants, but not in mammals. The FgILV5 deletion mutant ΔFgIlv5-4 failed to grow when the culture medium was nutritionally limited for BCAAs. When grown on potato-dextrose agar plates, ΔFgIlv5-4 exhibited a significant decrease in aerial hyphae formation and red pigmentation. Conidia formation was also blocked in ΔFgIlv5-4. Exogenous addition of 1 mM isoleucine and valine was able to rescue the defects of mycelial growth and conidial morphogenesis. Cellular stress assays showed that ΔFgIlv5-4 was more sensitive to osmotic and oxidative stresses than the wild-type strain. In addition, virulence of ΔFgIlv5-4 was dramatically reduced on wheat heads, and a low level of deoxynivalenol production was detected in ΔFgIlv5-4 in wheat kernels. The results of this study indicate that FgIlv5 is involved in valine and isoleucine biosynthesis and is required for full virulence in F. graminearum.

Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli.

2-methylpropan-1-ol (isobutanol) is a leading candidate biofuel for the replacement or supplementation of current fossil fuels. Recent work has demonstrated glucose to isobutanol conversion through a modified amino acid pathway in a recombinant organism. Although anaerobic conditions are required for an economically competitive process, only aerobic isobutanol production has been feasible due to an imbalance in cofactor utilization. Two of the pathway enzymes, ketol-acid reductoisomerase and alcohol dehydrogenase, require nicotinamide dinucleotide phosphate (NADPH); glycolysis, however, produces only nicotinamide dinucleotide (NADH). Here, we compare two solutions to this imbalance problem: (1) over-expression of pyridine nucleotide transhydrogenase PntAB and (2) construction of an NADH-dependent pathway, using engineered enzymes. We demonstrate that an NADH-dependent pathway enables anaerobic isobutanol production at 100% theoretical yield and at higher titer and productivity than both the NADPH-dependent pathway and transhydrogenase over-expressing strain. Our results show how engineering cofactor dependence can overcome a critical obstacle to next-generation biofuel commercialization.

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.

Factors enhancing L-valine production by the growth-limited L-isoleucine auxotrophic strain Corynebacterium glutamicum DeltailvA DeltapanB 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.

Interactions between large and small subunits of different acetohydroxyacid synthase isozymes of Escherichia coli.

The large, catalytic subunits (LSUs; ilvB, ilvG and ilvI, respectively) of enterobacterial acetohydroxyacid synthases isozymes (AHAS I, II and III) have molecular weights approximately 60 kDa and are paralogous with a family of other thiamin diphosphate dependent enzymes. The small, regulatory subunits (SSUs) of AHAS I and AHAS III (ilvN and ilvH) are required for valine inhibition, but ilvN and ilvH can only confer valine sensitivity on their own LSUs. AHAS II is valine resistant. The LSUs have only approximately 15, <<1 and approximately 3%, respectively, of the activity of their respective holoenzymes, but the holoenzymes can be reconstituted with complete recovery of activity. We have examined the activation of each of the LSUs by SSUs from different isozymes and ask to what extent such activation is specific; that is, is effective nonspecific interaction possible between LSUs and SSUs of different isozymes? To our surprise, the AHAS II SSU ilvM is able to activate the LSUs of all three of the isozymes, and the truncated AHAS III SSUs ilvH-Delta80, ilvH-Delta86 and ilvH-Delta89 are able to activate the LSUs of both AHAS I and AHAS III. However, none of the heterologously activated enzymes have any feedback sensitivity. Our results imply the existence of a common region in all three LSUs to which regulatory subunits may bind, as well as a similarity between the surfaces of ilvM and the other SSUs. This surface must be included within the N-terminal betaalphabetabetaalphabeta-domain of the SSUs, probably on the helical face of this domain. We suggest hypotheses for the mechanism of valine inhibition, and reject one involving induced dissociation of subunits.

Determination of rpoA as the most suitable internal control to study stress response in C. jejuni by RT-qPCR and application to oxidative stress.

Campylobacter jejuni represents one of the major causes of bacterial enteritis caused by food in humans. There are still mechanisms to be deciphered to better understand better its physiology and pathogenesis. Study of gene expression levels by RT-qPCR could be used, but to be accurate and reproducible, a good internal control has to be chosen. The aim of this study was to identify a highly stable housekeeping gene in Campylobacter jejuni that could constitute a good internal control to study gene expression variations between different growth phases or stress conditions. Expression levels of six different housekeeping genes (gyrA, ilvC, rpoA, slyD, thiC and rrs) were measured by RT-qPCR under different conditions (exponential phase, stationary phase, cold shock, cold shock+oxidative stress, oxidative stress). The rpoA gene was chosen as the best internal control. In a previous study, 9 proteins were identified as involved in oxidative stress response, among which 3 virulence factors. Expression levels of genes coding for these proteins was evaluated by RT-qPCR using rpoA as an internal control. The results obtained were concordant with what had been observed at the proteomic level, validating the methods used and confirming the hypothesis of a potential link between oxidative stress and virulence factors expression.

Crystal Structure and Biochemical Characterization of Ketol-Acid Reductoisomerase from Corynebacterium glutamicum.

l-Valine belongs to the branched-chain amino acids (BCAAs) and is an essential amino acid that is crucial for all living organisms. l-Valine is industrially produced by the nonpathogenic bacterium Corynebacterium glutamicum and is synthesized by the BCAA biosynthetic pathway. Ketol-acid reductoisomerase (KARI) is the second enzyme in the BCAA pathway and catalyzes the conversion of (S)-2-acetolactate into (R)-2,3-dihydroxy-isovalerate, or the conversion of (S)-2-aceto-2-hydroxybutyrate into (R)-2,3-dihydroxy-3-methylvalerate. To elucidate the enzymatic properties of KARI from C. glutamicum (CgKARI), we successfully produced CgKARI protein and determined its crystal structure in complex with NADP+ and two Mg2+ ions. Based on the complex structure, docking simulations, and site-directed mutagenesis experiments, we revealed that CgKARI belongs to Class I KARI and identified key residues involved in stabilization of the substrate, metal ions, and cofactor. Furthermore, we confirmed the difference in the binding of metal ions that depended on the conformational change.

Enhanced valine production in Corynebacterium glutamicum with defective H+-ATPase and C-terminal truncated acetohydroxyacid synthase.

We have reported increased glutamate production by a mutant of Corynebacterium glutamicum ATCC14067 (strain F172-8) with reduced H(+)-ATPase activity under biotin-limiting culture conditions (Aoki et al. Biosci. Biotechnol. Biochem., 69, 1466-1472 (2005)). In the present study, we examined valine production by an H(+)-ATPase-defective mutant of C. glutamicum. Using the double-crossover chromosome replacement technique, we constructed a newly defined H(+)-ATPase-defective mutant from ATCC13032. After transforming the new strain (A-1) with a C-terminal truncation of acetohydroxyacid synthase gene (ilvBN), valine production increased from 21.7 mM for the wild-type strain to 46.7 mM for the A-1 in shaking flask cultures with 555 mM glucose. Increased production of the valine intermediate acetoin was also observed in A-1, and was reduced by inserting acetohydroxyacid isomeroreductase gene (ilvC) into the ilvBN plasmid. After transformation with this new construct, valine production increased from 38.3 mM for the wild-type strain to 95.7 mM for A-1 strain. To the best of our knowledge, this is the first report indicating that an H(+)-ATPase-defective mutant of C. glutamicum is capable of valine production. Our combined results with glutamate and valine suggest that the H(+)-ATPase defect is also effective in the fermentative production of other practical compounds.

NADH/NADPH bi-cofactor-utilizing and thermoactive ketol-acid reductoisomerase from Sulfolobus acidocaldarius.

Ketol-acid reductoisomerase (KARI) is a bifunctional enzyme in the second step of branched-chain amino acids biosynthetic pathway. Most KARIs prefer NADPH as a cofactor. However, KARI with a preference for NADH is desirable in industrial applications including anaerobic fermentation for the production of branched-chain amino acids or biofuels. Here, we characterize a thermoacidophilic archaeal Sac-KARI from Sulfolobus acidocaldarius and present its crystal structure at a 1.75-Å resolution. By comparison with other holo-KARI structures, one sulphate ion is observed in each binding site for the 2'-phosphate of NADPH, implicating its NADPH preference. Sac-KARI has very high affinity for NADPH and NADH, with K M values of 0.4 µM for NADPH and 6.0 µM for NADH, suggesting that both are good cofactors at low concentrations although NADPH is favoured over NADH. Furthermore, Sac-KARI can catalyze 2(S)-acetolactate (2S-AL) with either cofactor from 25 to 60 °C, but the enzyme has higher activity by using NADPH. In addition, the catalytic activity of Sac-KARI increases significantly with elevated temperatures and reaches an optimum at 60 °C. Bi-cofactor utilization and the thermoactivity of Sac-KARI make it a potential candidate for use in metabolic engineering or industrial applications under anaerobic or harsh conditions.

Artificial domain duplication replicates evolutionary history of ketol-acid reductoisomerases.

The duplication of protein structural domains has been proposed as a common mechanism for the generation of new protein folds. A particularly interesting case is the class II ketol-acid reductoisomerase (KARI), which putatively arose from an ancestral class I KARI by duplication of the C-terminal domain and corresponding loss of obligate dimerization. As a result, the class II enzymes acquired a deeply embedded figure-of-eight knot. To test this evolutionary hypothesis we constructed a novel class II KARI by duplicating the C-terminal domain of a hyperthermostable class I KARI. The new protein is monomeric, as confirmed by gel filtration and X-ray crystallography, and has the deeply knotted class II KARI fold. Surprisingly, its catalytic activity is nearly unchanged from the parent KARI. This provides strong evidence in support of domain duplication as the mechanism for the evolution of the class II KARI fold and demonstrates the ability of domain duplication to generate topological novelty in a function-neutral manner.

The crystal structure of a bacterial class II ketol-acid reductoisomerase: domain conservation and evolution.

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes two steps in the biosynthesis of branched-chain amino acids. Amino acid sequence comparisons across species reveal that there are two types of this enzyme: a short form (Class I) found in fungi and most bacteria, and a long form (Class II) typical of plants. Crystal structures of each have been reported previously. However, some bacteria such as Escherichia coli possess a long form, where the amino acid sequence differs appreciably from that found in plants. Here, we report the crystal structure of the E. coli enzyme at 2.6 A resolution, the first three-dimensional structure of any bacterial Class II KARI. The enzyme consists of two domains, one with mixed alpha/beta structure, which is similar to that found in other pyridine nucleotide-dependent dehydrogenases. The second domain is mainly alpha-helical and shows strong evidence of internal duplication. Comparison of the active sites between KARI of E. coli, Pseudomonas aeruginosa, and spinach shows that most residues occupy conserved positions in the active site. E. coli KARI was crystallized as a tetramer, the likely biologically active unit. This contrasts with P. aeruginosa KARI, which forms a dodecamer, and spinach KARI, a dimer. In the E. coli KARI tetramer, a novel subunit-to-subunit interacting surface is formed by a symmetrical pair of bulbous protrusions.

Identification and optimization of a novel thermo- and solvent stable ketol-acid reductoisomerase for cell free isobutanol biosynthesis.

Due to its enhanced energy content and hydrophobicity, isobutanol is flagged as a next generation biofuel and chemical building block. For cellular and cell-free isobutanol production, NADH dependent (over NADPH dependent) enzyme systems are desired. To improve cell-free isobutanol processes, we characterized and catalytically optimized a NADH dependent, thermo- and solvent stable ketol-acid reductoisomerase (KARI) derived from the bacterium Meiothermus ruber (Mr). The wild type Mr-KARI has the most temperature tolerant KARI specific activity reported to date. The KARI screening procedure developed in this study allows accelerated molecular optimization. Thus, a KARI variant with a 350% improved activity and enhanced NADH cofactor specificity was identified. Other KARI variants gave insights into Mr-KARI structure-function relationships.

Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity.

The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol-acid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications.

NAD(P)H-hydrate dehydratase- a metabolic repair enzyme and its role in Bacillus subtilis stress adaptation.

BACKGROUND: One of the strategies for survival stress conditions in bacteria is a regulatory adaptive system called general stress response (GSR), which is dependent on the SigB transcription factor in Bacillus sp. The GSR is one of the largest regulon in Bacillus sp., including about 100 genes; however, most of the genes that show changes in expression during various stresses have not yet been characterized or assigned a biochemical function for the encoded proteins. Previously, we characterized the Bacillus subtilis168 osmosensitive mutant, defective in the yxkO gene (encoding a putative ribokinase), which was recently assigned in vitro as an ADP/ATP-dependent NAD(P)H-hydrate dehydratase and was demonstrated to belong to the SigB operon. METHODS AND RESULTS: We show the impact of YxkO on the activity of SigB-dependent Pctc promoter and adaptation to osmotic and ethanol stress and potassium limitation respectively. Using a 2DE approach, we compare the proteomes of WT and mutant strains grown under conditions of osmotic and ethanol stress. Both stresses led to changes in the protein level of enzymes that are involved in motility (flagellin), citrate cycle (isocitrate dehydrogenase, malate dehydrogenase), glycolysis (phosphoglycerate kinase), and decomposition of Amadori products (fructosamine-6-phosphate deglycase). Glutamine synthetase revealed a different pattern after osmotic stress. The patterns of enzymes for branched amino acid metabolism and cell wall synthesis (L-alanine dehydrogenase, aspartate-semialdehyde dehydrogenase, ketol-acid reductoisomerase) were altered after ethanol stress. CONCLUSION: We performed the first characterization of a Bacillus subtilis168 knock-out mutant in the yxkO gene that encodes a metabolite repair enzyme. We show that such enzymes could play a significant role in the survival of stressed cells.