Regulation of pigment content and enzyme activity in the cyanobacterium Nostoc sp. Mac grown in continuous light, a light-dark photoperiod, or darkness

AbstractBoth short-term and long-term adaptations of cyanobacterial metabolism to light and dark were studied in Nostoc sp. Mac. Long-term adaptations were induced by growing cells in the presence of glucose under (a) 30 μE m−2 s−1 continuous white light, (b) under a 14/10 h light/dark cycle, or (c) complete darkness. Short-term regulation of enzyme activities by light was then studied in cells rendered osmotically fragile with lysozyme. Cells were briefly illuminated then enzyme activities were measured following rapid lysis in a hypotonic assay medium. The following results were obtained. (1) Relative to fresh weight, dark-grown cells contained less chlorophyll, much less phycoerythrin, but similar amounts of phycocyanin compared to cells grown under either light regime. Relative to chlorophyll, the higher phycocyanin and much lower phycoerythrin in the dark-grown vs light-grown cells resembles long term changes in pigment content that occur during complementary chromatic adaptation to red vs orange light. (2) Both dark and light/dark grown cells displayed generally lowered photosynthetic activities compared to light-grown cells. The exception to this was the activity of fructose 1,6-bisphosphatase, which was higher in dark-grown cells. However, the photosynthetic induction period was markedly shorter in the light/dark-grown cells indicating an adaptation to changing illumination during growth. (3) Inhibitor studies using methyl viologen show that the fructose 1,6-bisphosphatase is reversibly light-activated in vivo by the cyanobacterial thioredoxin system under all growth conditions. Glucose-6-phosphate dehydrogenase activity was detected in cells grown in all conditions and this activity was reversibly deactivated by light or by dithiothreitol. In contrast, the protonmotive ATPase F0F1-type ATPase was fully active in both light and dark-adapted cells regardless of the light regime used for growth. (4) It is concluded that the Calvin cycle enzymes, their short-term regulatory system, including thioredoxin, glucose-6-phosphate dehydrogenase and an F0F1 ATPase not under thioredoxin control, are expressed in cells grown in complete darkness. Adaptation to heterotrophic growth in this cyanobacterium does not appear to involve synthesis of different enzyme forms lacking thioredoxin control sequences

Post-translational modification of heterologously expressed Streptomyces type II polyketide synthase acyl carrier proteins

AbstractExpression in Escherichia coli of Streptomyces acyl carrier proteins (ACPs) associated with polyketide biosynthesis using the pT7-7 expression system of Tabor and Richardson led to the production predominantly of inactive apo-proteins lacking the 4′-phosphopantetheinyl prosthetic group essential for polyketide synthase activity. Modification of growth conditions led to an increase of production of active holo-protein for the actinorhodin (act) ACP, but this technique was ineffective for oxytetracycline (otc) and griseusin (gris) ACPs. Labelling experiments revealed that a low level of otc ACP expressed prior to induction was produced mainly as active holo-protein, while post-induction 15N-labelled protein was almost exclusively in the apo-ACP form. Limiting endogenous holo-acyl carrier protein synthase (ACPS) concentration was implicated as responsible for low apo-ACP to holo-ACP conversion, rather than limiting substrate (coenzyme A) and cofactor (Mg2+) concentrations. Co-expression of act and gris ACPs with ACPS in E. coli led to high levels of production of active holo-ACPs and ACPS. We have also made the significant observation that ACPS is able to transfer acylated CoA moieties to act apo-ACP

Synthesis and evaluation of a (3R, 6S, 9S)-2-oxo-1-azabicyclo[4.3.0]nonane scaffold as a mimic of Xaa-transPro in poly-L-proline type II helix conformation

We describe the development of a small-molecule mimic of Xaa-trans-Pro dipeptide in poly-L-proline type II helix conformation, based upon a (3R,6S,9S)-2-oxo-1-azabicyclo[4.3.0]nonane core structure. Stereoselective synthesis of the mimic from L-pyroglutamic acid is achieved in twelve linear steps and 9.9% yield. Configurational and conformational analyses are conducted using a combination of 1H NMR spectroscopy, X-ray crystallography and circular dichroism spectroscopy; and evaluation of the mimic as a promising surrogate dipeptide, in a protein–protein interaction between the SH3 domain of human Fyn kinase (Fyn SH3) and peptidomimetics of its biological ligand, are conducted by 1H-15N HSQC NMR titration experiments

Acylation of Streptomyces type II polyketide synthase acyl carrier proteins

AbstractAcyl derivatives of type II PKS ACPs are required for in vitro studies of polyketide biosynthesis. The presence of an exposed cysteine residue prevented specific chemical acylation of the phosphopantetheine thiol of the actinorhodin PKS holo ACP. Acylation studies were further complicated by intramolecular disulphide formation between cysteine 17 and the phosphopantetheine. The presence of this intramolecular disulphide was confirmed by tryptic digestion of the ACP followed by ESMS analysis of the fragments. An act Cys17Ser ACP was engineered by site-directed mutagenesis. S-Acyl adducts of act C17S, oxytetracycline and griseusin holo ACPs were rapidly formed by reaction with hexanoyl, 5-ketohexanoyl and protected acetoacetyl imidazolides. Comparisons with type II FAS ACPs were made