羟氨苄青霉素引起大疱性类天疱疮样疹

1,4-Butanediol (BDO) is an important commodity chemical used to manufacture over 2.5 million tons annually of valuable polymers, and it is currently produced exclusively through feedstocks derived from oil and natural gas. Herein we report what are to our knowledge the first direct biocatalytic routes to BDO from renewable carbohydrate feedstocks, leading to a strain of Escherichia coli capable of producing 18 g l(-1) of this highly reduced, non-natural chemical. A pathway-identification algorithm elucidated multiple pathways for the biosynthesis of BDO from common metabolic intermediates. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic operation of the oxidative tricarboxylic acid cycle, thereby generating reducing power to drive the BDO pathway. The organism produced BDO from glucose, xylose, sucrose and biomass-derived mixed sugar streams. This work demonstrates a systems-based metabolic engineering approach to strain design and development that can enable new bioprocesses for commodity chemicals that are not naturally produced by living cells.

Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol

Activation of molecular oxygen by flavins and flavoproteins.

Cheese flavour formation by amino acid catabolism

The thermodynamic activity of proteins in solution is substantially altered by the addition of unreactive or 'inert' macromolecules occupying more than a few percent of total solution volume. Approximate theoretical models of this effect have been formulated using a simplified geometrical representation of molecular shapes. These models predict that under certain conditions, the structure and function of proteins in physiological media with a high total macromolecular content may be qualitatively different than in dilute solution. Experimental studies of the effect of 'inert' macromolecules on protein structure and/or function are reviewed, and it is found that under favorable circumstances the simplified models can provide a satisfactory semiquantitative description of the data.

The effect of volume occupancy upon the thermodynamic activity of proteins: some biochemical consequences

The effect of NADH on the activity of the purified pyruvate dehydrogenase complexes (PDHc) of Enterococcus (Ec.) faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli was determined in vitro. It was found that the PDHc of E. coli and L. lactis was active only at relatively low NADH/NAD ratios, whereas the PDHc of Ec. faecalis was inhibited only at high NADH/NAD ratios. The PDHc of Azotobacter vinelandii showed an intermediate sensitivity. The organisms were grown in chemostat culture under conditions that led to different intracellular NADH/NAD ratios and the PDHc activities in vivo could be calculated from the specific rates of product formation. Under anaerobic growth conditions, only Ec. faecelis expressed PDHc activity in vivo. The activities in vivo of the complexes of the different organisms were in good agreement with their properties determined in vitro. The physiological consequences of these results are discussed.

Differences in sensitivity to NADH of purified pyruvate dehydrogenase complexes of Enterococcus faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli: Implications for their activity in vivo

Labelling studies with N-ethylmaleimide show that either in the presence of Mg2+, thiamine pyrophosphate (TPP) and pyruvate or in the presence of NADH the overall activity of the pyruvate dehydrogenase complex from Azotobacter vinelandii is inhibited without much inhibition of the partial reactions. The complex undergoes a conformational change upon incubation with NADH. The inhibition by bromopyruvate is less specific.



Specific incorporation of a fluorescent maleimide derivative was observed on the two transacetylase isoenzymes. Binding studies with a similar spin label analogue show that 3 molecules/FAD are incorporated by incubation of pyruvate, Mg2+ and TPP, whereas 2 molecules/FAD are incorporated via incubation with NADH. The spin label spectra support the idea that in the complex the active centres of the component enzymes are connected by rapid rotation of the lipoyl moiety.



Three acetyl groups are incorporated in the complex by incubation with [2-14C]pyruvate. Time-dependent incorporation supports the view that the two transacetylase isoenzymes react in non-identical ways with the pyruvate dehydrogenase components of the complex. The results show that the complex contains 2 low-molecular-weight transacetylase molecules and 4 molecules of the high-molecular-weight isoenzyme.



Mn2+-binding studies show that the complex binds 10 ions, with different affinities. 2 Mn2+ ions are bound with a 20-fold higher affinity than the remaining 8 Mn2+ ions. The latter 8 ions bind with equal affinities and are thought to reflect binding to the pyruvate dehydrogenase components of the complex.



It is concluded that the complex contains 8 pyruvate dehydrogenase molecules, 4 high-molecular-weight transacetylase molecules, 2 low-molecular-weight transacetylase molecules and 1 dimeric (2-FAD-containing) symmetric molecule of lipoamide dehydrogenase. Evidence comes from pyruvate-dependent inactivation and labelling studies that the pyruvate dehydrogenase components contain either an –SH group or an S-S bridge which participates in the hydroxyethyl transfer to the transacetylase components.

The Pyruvate‐Dehydrogenase Complex from Azotobacter vinelandii

The gene encoding p-hydroxybenzoate hydroxylase from Pseudomonas,fluorescens was cloned in Escherichia coli to provide DNA for mutagenesis studies on thc protein product. A plasmid containing a 1.65-kbp insert of P. fluorescens chromosomal DNA was obtained and its nucleotide sequence determined. The DNA-derived amino acid sequence agrees completely with the chemically determined amino acid sequence of the isolated protein. The enzyme is strongly expressed under influence of the vector-encoded lac promotor and is purified to homogeneity in a simple three-step procedure. The relation between substrate binding, the effector role of substrate and hydroxylation efficiency was studied by use of site-directed rnutagenesis. Arg214, in ion-pair interaction with the carboxy moiety of p-hydroxybenzoate, was replaced with Lys, Gln and Ala, respectively. The affinity of the free enzymes for NADPH is unchanged, whereas the affinity for the aromatic substrate is strongly decreased. For enzymes Arg214jAla and Arg214+Gln, the effector role of substrate is lost. For enzyme Arg214+Lys, binding of p-hydroxybenzoate highly stimulates the rate of flavin reduction. In the presence of substrate or substrate analogues, the reduced enzyme Arg214jLys fails to stabilize the 4a-hydroperoxyflavin intermediate, essential for efficient hydroxylation. Like the wild-type, enzyme Arg214+Lys is susceptible to substrate inhibition. From spectral and kinetic results it is suggested that secondary binding of the substrate occurs at the re side of the flavin, where the nicotinamide moiety of NADPH is supposed to bind.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1992.tb17436.x

Substitution of Arg214 at the substrate-binding site of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

Regulation of dinitrogen fixation in intact Azotobacter vinelandii.

Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced with other residues. His450, the active-site base, was replaced with Ser, Tyr or Phe. Pro451, from X-ray analysis found to be in cis conformation positioning the backbone carbonyl of His450 close to N3 of the flavin, was changed to Ala. Glu455, from X-ray analysis expected to be involved in modulating the pK, of the base (His450), was replaced with Asp and Gln. The general conclusion is that mutation of the His-Glu diad impairs intramolecular electron transfer between the disulfide/dithiol and the FADH-/FAD. The wild-type enzyme functions according to a ping-pong mechanism in the physiological reaction in which the formation of NADH is rate-limiting. Above pH 8.0 the enzyme is strongly inhibited by the product NADH. The pH dependence of the steady-state kinetics using the NAD' analog 3acetylpyridine adenine dinucleotide (AcPyAde') reveals a pK, of 8.1 in the pK, AcPyAde+ plot indicating that this pK, is related to the deprotonation of His450 [Benen, J., Berkel van, W., Zak, Z., Visser, T., Veeger, C. & Kok de, A. (1991) Eur. J. Biochem. 202, 863-8721 and to the inhibition by NADH. The mutations considerably affect turnover. Enzymes with the mutations Pro451 + Ala, His450 + Phe and His450 + Tyr appear to be almost inactive in both directions. Enzyme His450 + Ser is minimally active, V at the pH optimum being 0.5% of wild-type activity in the physiological reaction. Rapid reaction kinetics show that for the His450-mutated enzymes the reductive half reaction using reduced 6,8-thioctic acid amide [Lip(SH),] is rate-limiting and extremely slow when compared to the wild-type enzyme. For enzyme Pro451 + Ala it is concluded that the loss of activity is due to over-reduction by Lip(SH)2 and NADH. The Glu455-mutated enzymes are catalytically competent but show strong inhibition by the product NADH (enzyme Glu455 + Asp more than Glu455 --t Gln). The inhibition can largely be overcome by using AcPyAde' instead of NAD' in the physiological reaction. The rapid reaction kinetics obtained for enzymes Glu455 +Asp and Glu455hGln deviate from the wild-type enzyme. It is concluded that this difference is due to cooperativity between the active sites in this dimeric enzyme. Rapid reaction kinetics of enzymes His450 + Ser and Glu455 + Gln show the existence of two intermediates at the two-electron reduced level: a species with the NAD' bound, the flavin reduced and the disulfide intact (oxidized) and a species with NAD' bound, the disulfide reduced and the flavin oxidized. No spectral evidence is obtained for the participation of a proposed flavin C4a adduct intermediate [Thorpe, C. & Williams, C. H. Jr (1981) Biochemistry 20, 1507- 15131 in the reaction mechanism of enzymes His450 + Ser and Glu455 + Gln.

/pdf/lipoamide-dehydrogenase-from-azotobacter-vinelandii-site-5f3mrq9j58.pdf

Lipoamide dehydrogenase from Azotobacter vinelandii: site directed mutagenesis of the his450-glu455 diad. Spectral properties of wild type and mutated enzymes.

Arie de Kok

Papers

Differences in sensitivity to NADH of purified pyruvate dehydrogenase complexes of Enterococcus faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli: Implications for their activity in vivo

The Pyruvate‐Dehydrogenase Complex from Azotobacter vinelandii

Substitution of Arg214 at the substrate-binding site of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

Regulation of dinitrogen fixation in intact Azotobacter vinelandii.

Lipoamide dehydrogenase from Azotobacter vinelandii: site directed mutagenesis of the his450-glu455 diad. Spectral properties of wild type and mutated enzymes.