Overexpression of NAD kinases improves the L-isoleucine biosynthesis in Corynebacterium glutamicum ssp. lactofermentum

NADPH is the key cofactor in L-isoleucine (Ile) biosynthetic pathway. To increase the Ile biosynthesis in Corynebacterium glutamicum ssp. lactofermentum JHI3-156, NADPH supply needs to be enhanced. Here NAD kinase, the key enzyme for the de novo biosynthesis of NADP(+) and NADPH, were cloned and expressed in JHI3-156, and their influences on Ile production were analysed. Meanwhile, enzyme properties of NAD kinase from JHI3-156 (CljPpnK) were compared with that from C. glutamicum ssp. lactofermentum ATCC 13869 (ClPpnK). Four variations existed between CljPpnK and ClPpnK. Both PpnKs were poly(P)/ATP-dependent NAD kinases that used ATP as the preferred phosphoryl donor and NAD(+) as the preferred acceptor. CljPpnK exhibited a higher activity and stability than ClPpnK and less sensitivity towards the effectors NADPH, NADP(+), and NADH, partly due to the variations between them. The S57P variation decreased their activity. Expression of CljppnK and ClppnK in JHI3-156 increased the ATP-NAD(+) kinase activity by 69- and 47-fold, respectively, the intracellular NADP(+) concentration by 36% and 101%, respectively, the NADPH concentration by 95% and 42%, respectively, and Ile production by 37% and 24%, respectively. These results suggest that overexpressing NAD kinase is a useful metabolic engineering strategy to improve NADPH supply and isoleucine biosynthesis.     

Biphasic Kinetic Behavior of E. coli WrbA, an FMN-Dependent NAD(P)H:Quinone Oxidoreductase

The E. coli protein WrbA is an FMN-dependent NAD(P)H:quinone oxidoreductase that has been implicated in oxidative defense. Three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds. The present work was undertaken to evaluate these suggestions and to characterize the kinetic behavior of WrbA. Steady-state kinetics results reveal that WrbA conforms to a ping-pong mechanism with respect to the constancy of the apparent Vmax to Km ratio with substrate concentration. However, the competitive/non-competitive patterns of product inhibition, though consistent with the general class of bi-substrate reactions, do not exclude a minor contribution from additional forms of the enzyme. NMR results support the presence of additional enzyme forms. Docking and energy calculations find that electron-transfer-competent binding sites for NADH and benzoquinone present severe steric overlap, consistent with the ping-pong mechanism. Unexpectedly, plots of initial velocity as a function of either NADH or benzoquinone concentration present one or two Michaelis-Menten phases depending on the temperature at which the enzyme is held prior to assay. The effect of temperature is reversible, suggesting an intramolecular conformational process. WrbA shares these and other details of its kinetic behavior with mammalian DT-diaphorase, an FAD-dependent NAD(P)H:quinone oxidoreductase. An extensive literature review reveals several other enzymes with two-plateau kinetic plots, but in no case has a molecular explanation been elucidated. Preliminary sedimentation velocity analysis of WrbA indicates a large shift in size of the multimer with temperature, suggesting that subunit assembly coupled to substrate binding may underlie the two-plateau behavior. An additional aim of this report is to bring under wider attention the apparently widespread phenomenon of two-plateau Michaelis-Menten plots.     

聚乙烯醇-明胶复合膜固定化重组大肠杆菌NAD激酶的研究

为提高烟酰胺腺嘌呤二核苷酸(NAD)激酶的稳定性,采用复合膜对NAD激酶进行固定化研究。选用聚乙烯醇(PVA)、聚乳酸(PLA)、海藻酸钠(SA)和明胶(GEL)膜材料固定化NAD激酶。通过单因素实验确定最佳固定化条件为:PVA∶GEL为4∶1,加酶量为0.6 mL,固定化时间为6h,固定化温度为35℃,此时酶活力回收率达到最高值84%。固定化酶酶学性质分析结果表明,与游离酶进行比较,固定化后NAD激酶的最适温度由50℃提高至55℃,最适pH由8.0降至7.0,NAD激酶的热稳定性和pH稳定性均得到显著提高,但固定化酶的亲和力降低。固定化NAD激酶重复利用6次后,酶活性依然可维持初始酶活性的75%以上,表明聚乙烯醇-明胶复合膜固定化酶具有良好的操作稳定性。     

Structure of the adenylation domain of an NAD+-dependent DNA ligase.

BACKGROUND: DNA ligases catalyse phosphodiester bond formation between adjacent bases in nicked DNA, thereby sealing the nick. A key step in the catalytic mechanism is the formation of an adenylated DNA intermediate. The adenyl group is derived from either ATP (in eucaryotes and archaea) or NAD+4 (in bacteria). This difference in cofactor specificity suggests that DNA ligase may be a useful antibiotic target. RESULTS: The crystal structure of the adenylation domain of the NAD+-dependent DNA ligase from Bacillus stearothermophilus has been determined at 2.8 A resolution. Despite a complete lack of detectable sequence similarity, the fold of the central core of this domain shares homology with the equivalent region of ATP-dependent DNA ligases, providing strong evidence for the location of the NAD+-binding site. CONCLUSIONS: Comparison of the structure of the NAD+4-dependent DNA ligase with that of ATP-dependent ligases and mRNA-capping enzymes demonstrates the manifold utilisation of a conserved nucleotidyltransferase domain within this family of enzymes. Whilst this conserved core domain retains a common mode of nucleotide binding and activation, it is the additional domains at the N terminus and/or the C terminus that provide the alternative specificities and functionalities in the different members of this enzyme superfamily.     

K+-dependent enzyme activity of H+-ATPase in the E. coli membrane

It was shown that DCCD-sensitive ATPase activity of isolated membranes and preparations of F1F0 only from anaerobically grown E. coli depended on K+ activity. F1F0 include two additional proteins which correspond to the Trk system. The data improve the possibility to form supercomplex (F1F0-Trk) functioning as the H(+)-K(+)-pump.     

Expression of the Escherichia coli catabolic threonine dehydratase in Corynebacterium glutamicum and its effect on isoleucine production.

The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1. 16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to alpha-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by the ilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and a tdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.     

Identity of Escherichia coli D-1-amino-2-propanol:NAD+ oxidoreductase with E. coli glycerol dehydrogenase but not with Neisseria gonorrhoeae 1,2-propanediol:NAD+ oxidoreductase.

The properties of D-1-amino-2-propanol oxidoreductase from wild-type Escherichia coli have been compared with those of a glycerol dehydrogenase from mutant E. coli 424 and of a 1,2-propanediol oxidoreductase from Neisseria gonorrhoeae. Several independent lines of evidence indicate that the former two enzymes are identical. (i) Both enzymatic activities purified to virtual homogeneity in an identical manner, and the ratio of specific activities (glycerol/aminopropanol) remained constant at all stages. (ii) When electrophoresed, both purified enzymes showed a major as well as a minor band of protein coincident with activity, and these two bands from each enzyme had the same mobility. (iii) The subunit molecular weights and isoelectric points were identical for each enzyme, and (iv) kinetic constants (Km and Vmax values) determined with three different substrates were the same. The somewhat greater stability of the glycerol dehydrogenase to controlled heat denaturation at 74 degrees C was the only difference observed between these two enzymes. In contrast, D-1-amino-2-propanol oxidoreductase was found to be immunochemically and kinetically distinct from the 1,2-propanediol oxidoreductase from N. gonorrhoeae.     

Expression of alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli and molecular characterization of the recombinant alanine racemase

We constructed the high-expression system of the alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli BL 21 (DE3) to characterize the enzymological and structural properties of the gene product, Alr. The Alr was expressed in the soluble fractions of the cell extract of the E. coli clone and showed alanine racemase activity. The purified Alr was a dimer with a molecular mass of 78 kDa. The Alr required pyridoxal 5'-phosphate (PLP) as a coenzyme and contained 2 mol of PLP per mol of the enzyme. The holoenzyme showed maximum absorption at 420 nm, while the reduced form of the enzyme showed it at 310 nm. The Alr was specific for alanine, and the optimum pH was observed at about nine. The Alr was relatively thermostable, and its half-life time at 60 degrees C was estimated to be 26 min. The K(m) and V(max) values were determined as follows: l-alanine to d-alanine, K(m) (l-alanine) 5.01 mM and V(max) 306 U/mg; d-alanine to l-alanine, K(m) (d-alanine) 5.24 mM and V(max) 345 U/mg. The K(eq) value was calculated to be 1.07 and showed good agreement with the theoretical value for the racemization reaction. The high substrate specificity of the Alr from C. glutamicum ATCC 13032 is expected to be a biocatalyst for d-alanine production from the l-counter part.     

Chemistry of gene silencing: the mechanism of NAD+-dependent deacetylation reactions.

The Sir2 enzyme family is responsible for a newly classified chemical reaction, NAD(+)-dependent protein deacetylation. New peptide substrates, the reaction mechanism, and the products of the acetyl transfer to NAD(+) are described for SIR2. The final products of SIR2 reactions are the deacetylated peptide and the 2' and 3' regioisomers of O-acetyl ADP ribose (AADPR), formed through an alpha-1'-acetyl ADP ribose intermediate and intramolecular transesterification reactions (2' --> 3'). The regioisomers, their anomeric forms, the interconversion rates, and the reaction equilibria were characterized by NMR, HPLC, 18O exchange, and MS methods. The mechanism of acetyl transfer to NAD(+) includes (1) ADP ribosylation of the peptide acyl oxygen to form a high-energy O-alkyl amidate intermediate, (2) attack of the 2'-OH group on the amidate to form a 1',2'-acyloxonium species, (3) hydrolysis to 2'-AADPR by the attack of water on the carbonyl carbon, and (4) an SIR2-independent transesterification equilibrating the 2'- and 3'-AADPRs. This mechanism is unprecedented in ADP-ribosyl transferase enzymology. The 2'- and 3'-AADPR products are candidate molecules for SIR2-initiated signaling pathways.     

Overexpression of ppc and lysC to improve the production of 4-hydroxyisoleucine and its precursor l-isoleucine in recombinant Corynebacterium glutamicum ssp. lactofermentum

4-hydroxyisoleucine (4-HIL) exhibits unique insulinotropic and insulin-sensitizing activities and is an attractive candidate for the treatment of type II and type I diabetes. In our previous study, l-isoleucine dioxygenase gene (ido) was cloned and overexpressed in an l-isoleucine-producing strain, Corynebacterium glutamicum ssp. lactofermentum SN01, and 4-HIL was produced from the endogenous l-isoleucine (Ile). In this study, ppc and lysC were co-expressed with ido to increase the supply of Ile, the direct precursor of 4-HIL, and to further improve the 4-HIL yield. After 144 h of fermentation, the ido-ppc-expressing strain produced 95.72 ± 1.52 mM 4-HIL, 29% higher than the ido-expressing strain. The co-expression of lysC and ppc with ido resulted in a further 35% increment of carbon flux to l-aspartate family amino acids biosynthesis pathway. However, the conversion ratio of Ile to 4-HIL and the 4-HIL yield decreased to 0.31 mol/mol and 30.16 ± 2.01 mM, respectively, likely due to the decreased IDO activity caused by lower pH and higher intracellular Ile concentration. Therefore, co-expression of ido and ppc was benefit for 4-HIL de novo biosynthesis, while co-expression of lysC with ido and ppc decreased the conversion ratio of Ile to 4-HIL.     

Increased recombinant frequencies with an amber mutation in the gal-operon of E. coli

Summary Two closely linked mutations, U20 and M87, in the deletion group V. of the transferase gene of the galactose operon in E. coli were crossed against a set of other mutations in the epimerase, transferase and kinase genes of the galactose operon by P1 transduction. U20 is an amber mutation. Its Gal ⁺ frequencies are higher by a factor of 2 to 10 than those of M87. Two double mutants of U20 with other gal mutations do not show these Gal ⁺ frequencies in similar crosses. The results can be explained on the assumption that in heteroduplexes U20 is repaired to yield wildtype at higher rate than is M87.     

Production of succinic acid through overexpression of NAD(+)-dependent malic enzyme in an Escherichia coli mutant.

NAD(+)-dependent malic enzyme was cloned from the Escherichia coli genome by PCR based on the published partial sequence of the gene. The enzyme was overexpressed and purified to near homogeneity in two chromatographic steps and was analyzed kinetically in the forward and reverse directions. The Km values determined in the presence of saturating cofactor and manganese ion were 0.26 mM for malate (physiological direction) and 16 mM for pyruvate (reverse direction). When malic enzyme was induced under appropriate culture conditions in a strain of E. coli that was unable to ferment glucose and accumulated pyruvate, fermentative metabolism of glucose was restored. Succinic acid was the major fermentation product formed. When this fermentation was performed in the presence of hydrogen, the yield of succinic acid increased. The constructed pathway represents an alternative metabolic route for the fermentative production of dicarboxylic acids from renewable feedstocks.