INVESTIGATIONS ON THE INTRACELLULAR LOCALIZATION OF PHOSPHATIDYLSERINE SYNTHASE FROM ESCHERICHIA COLI

Phosphatidylserine synthase catalyzes the committed step in the synthesis of the major lipid of Escherichia coli, phosphatidylethanolamine, and may be involved in regulating the balance of the zwitterionic and anionic phospholipids in the membrane. Unlike the other enzymes involved in the biosynthesis of phospholipids in E. coli, phosphatidylserine synthase is not membrane associated but seems to have a high affinity for the ribosomal fraction of cells broken by various methods. Investigations on the enzyme in cell free extracts using glycerol gradient centrifugation revealed that the binding of the synthase to ribosomes may be prevented by the presence of highly basic compounds such as spermidine and by the presence of detergent-lipid substrate micelles under assay conditions. Thus phosphatidylserine synthase may not be ribosome associated under physiological conditions but associated with its membrane bound substrate (Louie and Dowhan (1980) J. Biol. Chem. 255, 1124).^ In addition homogeneous enzyme shows many of the properties of a membrane associated protein. It binds nonionic detergent such as Triton X-100, which is also required during purification of the enzyme. Optimal catalytic activity is also dependent on micelle or surface bound substrate. Phosphatidylserine synthase has been synthesized in vitro using a coupled transcription-translation system dependent on the presence of the cloned structural gene. The translation product was found to preferentially associate with the ribosomal fraction even in the presence of added E. coli membranes. Preferential membrane binding could be induced if the membranes were supplemented with the lipid substrate CDP-diacylglycerol. Similar effects were obtained with the acidic lipids phosphatidylglycerol and cardiolipin. On the other hand the zwitterionic lipid phosphatidylethanolamine and the lipid product phosphatidylserine did not cause any detectable membrane association. These results are consistent with the enzyme recognizing membrane bound substrate (Carman and Dowhan (1979) J. Biol. Chem. 254, 8391) and with the lipid charge influencing membrane interaction.^ Phosphatidylserine synthase is at a branch point in lipid metabolism, which may determine the distribution of the zwitterionic and anionic phospholipids in the membrane. The results obtained here indicate phosphatidylserine synthase may play a significant role in membrane lipid biosynthesis by maintaining charge balance of the E. coli membrane. In determining the localization of phosphatidylserine synthase in vitro one may have a better understanding of its function and control in vivo and may also have a better understanding of its role in membrane assembly.^

THE EQUILIBRIUM CONSTANT FOR THE GLYCINE SYNTHASE REACTION (ONE-CARBON, METABOLISM, FOLATE)

The equilibrium constant (K(,c)) under physiological conditions (38(DEGREES)C, 0.25 M ionic strength (I), pH 7.0) for the glycine synthase (GS) reaction (E C 2.1.2.1.0) (Equation 1) has been determined. (UNFORMATTED TABLE FOLLOWS)^ 5,10-CH(,2)-H(,4)Folate NADH NH (,4)+ CO(,2) ^ K(,c) = Eq. 1^ H(,4)Folate NAD('+) GLY ^(TABLE ENDS)^ The enzymatic instability of the GS enzyme complex itself has made it necessary to determine the overall K(,c) from the product of constants for the partial reactions of GS determined separately under the same conditions. The partial reactions are the H(,4)Folate-formaldehyde (CH(,2)(OH)(,2)) condensation reaction (Reaction 1) the K(,c) for which has been reported by this laboratory (3.0 x 10('4)), the lipoate (LipS(,2)) dehydrogenase reaction (LipDH) (Reaction 2) and the Gly-Lip^ decarboxylase reaction (Reaction 3) forming reduced lipoate (Lip(SH)(,2)), NH(,4)('+), CO(,2) and CH(,2)(OH)(,2.) (UNFORMATTED TABLE FOLLOWS)(,)^ H(,4)Fote + CH(,2)(OH)(,2) 5,10-CH(,2)-H(,4)Folate (1)^ Lip(SH)(,2) + NAD('+) LipS(,2) + NADH + H('+) (2)^ H('+) + Gly + LipS(,2) Lip(SH)(,2) + NH(,4)('+) CO(,2) + CH(,2)(OH)(,2) (3)^(TABLE ENDS)^ In this work the K(,c) for Reactions 2 and 3 are reported.^ The K(,c)' for the LipDH reaction described by other authors was reported with unexplainable conclusions regarding the pH depend- ence for the reaction. These conclusions would imply otherwise unexpected acid dissociation constants for reduced and oxidized lipoate. The pK(,a)',s for these compounds have been determined to resolve discrepancy. The conclusions are as follows: (1) The K(,c) for the LipDH reaction is 2.08 x 10('-8); (2) The pK(,a)',s for Lip(SH)(,2) are 4.77(-COOH), 9.91(-SH), 11.59(-SH); for LipS(,2) the carboxyl pK(,a)' is 4.77; (3) Contrary to previous literature, the log K(,c)' for the LipDH reaction is a linear function of the pH, a conclusion supported by the values for the dissociation constants.^ The K(,c) for Reaction 3 is the product of constants for Reactions 4-7. (UNFORMATTED TABLE FOLLOWS)^ LipSHSCH(,2)OH + H(,2)O Lip(SH)(,2) + CH(,2)(OH)(,2) (4)^ H(,2)O + LipSHSCH(,2)NH(,3)('+) LipSHSCH(,2)OH + NH(,4)('+) (5)^ LipSHSCH(,2)NH(,2) + H('+) LipSHSCH(,2)NH(,3)('+) (6)^ Gly + LipS(,2) LipSHSCH(,2)NH(,2) + CO(,2) (7)^(TABLE ENDS)^ Reactions 4-6 are non-enzymatic reactions whose constants were determined spectrophotometrically. Reaction 7 was catalyzed by the partially purified P-protein of GS with equilibrium approached from both directions. The value for K(,c) for this reaction is 8.15 x 10('-3). The combined K(,c) for Reactions 4-7 or Reaction 3 is 2.4 M.^ The overall K(,c) for the GS reaction determined by combination of values for Reactions 1-3 is 1.56 x 10('-3). ^

The effect of specific carotenoids on lung cancer risk

A population-based case-comparison study of histologically confirmed lung cancer among white male and female residents of six Texas coastal counties was conducted from 1979-1982. Dietary information as well as information concerning smoking, alcohol consumption, occupational and residential exposures, and family history of cancer was obtained from 149 living cases and 359 comparison subjects.^ These data support the findings of previous studies that reported a protective association of total carotene intake with lung cancer (OR = 4.07, CI = 3.36-4.78), and no association for total Vitamin A or retinol. Of six specific carotenoids examined, these data reveal a statistically significant protective effect for alpha carotene (OR = 3.58, CI = 2.85-4.31), and an elevated, although non-significant effect for beta carotene (OR = 1.46, CI = 0.61-2.31). No consistent significant effect was found for cryptoxanthin, other xanthins, lutein or lycopene.^ Similar results were found for both males and females, and for both squamous cell and adenocarcinoma subtypes, although loss of power resulting from stratification may have rendered the celltype specific results more imprecise. These results should be considered with caution until confirmed by other studies, however, they suggest the importance of evaluating specific carotenoids in future diet-lung cancer investigations. ^

(Table 1) Chlorophyll concentration and pigment composition of carotenoids in three heathland bryophytes, northern Sweden

Background and Aims: Anthropogenic depletion of stratospheric ozone in Arctic latitudes has resulted in an increase of ultraviolet-B radiation (UV-B) reaching the biosphere. UV-B exposure is known to reduce aboveground biomass and plant height, to increase DNA damage and cause accumulation of UV-absorbing compounds in polar plants. However, many studies on Arctic mosses tended to be inconclusive. The importance of different water availability in influencing UV-B impacts on lower plants in the Arctic has been poorly explored and might partially explain the observed wide variation of responses, given the importance of water in controlling bryophyte physiology. This study aimed to assess the long-term responses of three common sub-Arctic bryophytes to enhanced UV-B radiation (+UV-B) and to elucidate the influence of water supply on those responses. Results: Responses were species specific: H. splendens responded most to +UV-B, with reduction in both annual growth (-22%) and sporophyte production (-44%), together with increased b-carotene, violaxanthin, total chlorophyll and NPQ, and decreased zeaxanthin and de-epoxidation of the xanthophyll cycle pool (DES). Barbilophozia lycopodioides responded less to +UV-B, showing increased b-carotene and sclerophylly and decreased UV-absorbing compounds. Polytrichum commune only showed small morphogenetic changes. No effect of UV-B on bryophyte cover was observed. Water availability had profound effects on bryophyte ecophysiology, and plants showed, in general, lower growth and ETR, together with a higher photoprotection in the drier site. Water availability also influenced bryophyte responses to +UV-B and, in particular, responses were less detectable in the drier site. Conclusions: Impacts of UV-B exposure on Arctic bryophytes were significant, in contrast to modest or absent UV-B effects measured in previous studies. The impacts were more easily detectable in species with high plasticity such as H. splendens and less obvious, or more subtle, under drier conditions. Species biology and water supply greatly influences the impact of UV-B on at least some Arctic bryophytes and could contribute to the wide variation of responses observed previously.

Ectopic expression of the minimal whiE polyketide synthase generates a library of aromatic polyketides of diverse sizes and shapes

The single recombinant expressing the Streptomyces coelicolor minimal whiE (spore pigment) polyketide synthase (PKS) is uniquely capable of generating a large array of well more than 30 polyketides, many of which, so far, are novel to this recombinant. The characterized polyketides represent a diverse set of molecules that differ in size (chain length) and shape (cyclization pattern). This combinatorial biosynthetic library is, by far, the largest and most complex of its kind described to date and indicates that the minimal whiE PKS does not independently control polyketide chain length nor dictate the first cyclization event. Rather, the minimal PKS enzyme complex must rely on the stabilizing effects of additional subunits (i.e., the cyclase whiE-ORFVI) to ensure that the chain reaches the full 24 carbons and cyclizes correctly. This dramatic loss of control implies that the growing polyketide chain does not remain enzyme bound, resulting in the spontaneous cyclization of the methyl terminus. Among the six characterized dodecaketides, four different first-ring cyclization regiochemistries are represented, including C7/C12, C8/C13, C10/C15, and C13/C15. The dodecaketide TW93h possesses a unique 2,4-dioxaadamantane ring system and represents a new structural class of polyketides with no related structures isolated from natural or engineered organisms, thus supporting the claim that engineered biosynthesis is capable of producing novel chemotypes.

Regulation of p53 expression by thymidylate synthase

Previous studies showed that thymidylate synthase (TS), as an RNA binding protein, regulates its own synthesis by impairing the translation of TS mRNA. In this report, we present evidence that p53 expression is affected in a similar manner by TS. For these studies, we used a TS-depleted human colon cancer HCT-C cell that had been transfected with either the human TS cDNA or the Escherichia coli TS gene. The level of p53 protein in transfected cells overexpressing human TS was significantly reduced when compared with its corresponding parent HCT-C cells. This suppression of p53 expression was the direct result of decreased translational efficiency of p53 mRNA. Similar results were obtained upon transfection of HCT-C cells with pcDNA 3.1 (+) containing the E. coli TS gene. These findings provide evidence that TS, from diverse species, specifically regulates p53 expression at the translational level. In addition, TS-overexpressing cells with suppressed levels of p53 are significantly impaired in their ability to arrest in G1 phase in response to exposure to a DNA-damaging agent such as γ-irradiation. These studies provide support for the in vivo biological relevance of the interaction between TS and p53 mRNA and identify a molecular pathway for controlling p53 expression.

Control of phosphatidylserine biosynthesis through phosphatidylserine-mediated inhibition of phosphatidylserine synthase I in Chinese hamster ovary cells

Phosphatidylserine (PtdSer) synthesis in Chinese hamster ovary (CHO) cells occurs through the exchange of l-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine. The synthesis is depressed on the addition of PtdSer to the culture medium. A CHO cell mutant named mutant 29, whose PtdSer biosynthesis is highly resistant to this depression by exogenous PtdSer, has been isolated from CHO-K1 cells. In the present study, the PtdSer-resistant PtdSer biosynthesis in the mutant was traced to a point mutation in the PtdSer synthase I gene, pssA, resulting in the replacement of Arg-95 of the synthase by lysine. Introduction of the mutant pssA cDNA, but not the wild-type pssA cDNA, into CHO-K1 cells induced the PtdSer-resistant PtdSer biosynthesis. In a cell-free system, the serine base-exchange activity of the wild-type pssA-transfected cells was inhibited by PtdSer, but that of the mutant pssA-transfected cells was resistant to the inhibition. Like the mutant 29 cells, the mutant pssA-transfected cells grown without exogenous PtdSer exhibited an ≈2-fold increase in the cellular PtdSer level compared with that in CHO-K1 cells, although the wild-type pssA-transfected cells did not exhibit such a significant increase. These results indicated that the inhibition of PtdSer synthase I by PtdSer is essential for the maintenance of a normal PtdSer level in CHO-K1 cells and that Arg-95 of the synthase is a crucial residue for the inhibition.

Interacting helical faces of subunits a and c in the F1Fo ATP synthase of Escherichia coli defined by disulfide cross-linking

Subunits a and c of Fo are thought to cooperatively catalyze proton translocation during ATP synthesis by the Escherichia coli F1Fo ATP synthase. Optimizing mutations in subunit a at residues A217, I221, and L224 improves the partial function of the cA24D/cD61G double mutant and, on this basis, these three residues were proposed to lie on one face of a transmembrane helix of subunit a, which then interacted with the transmembrane helix of subunit c anchoring the essential aspartyl group. To test this model, in the present work Cys residues were introduced into the second transmembrane helix of subunit c and the predicted fourth transmembrane helix of subunit a. After treating the membrane vesicles of these mutants with Cu(1,10-phenanthroline)2SO4 at 0°, 10°, or 20°C, strong a–c dimer formation was observed at all three temperatures in membranes of 7 of the 65 double mutants constructed, i.e., in the aS207C/cI55C, aN214C/cA62C, aN214C/cM65C, aI221C/cG69C, aI223C/cL72C, aL224C/cY73C, and aI225C/cY73C double mutant proteins. The pattern of cross-linking aligns the helices in a parallel fashion over a span of 19 residues with the aN214C residue lying close to the cA62C and cM65C residues in the middle of the membrane. Lesser a–c dimer formation was observed in nine other double mutants after treatment at 20°C in a pattern generally supporting that indicated by the seven landmark residues cited above. Cross-link formation was not observed between helix-1 of subunit c and helix-4 of subunit a in 19 additional combinations of doubly Cys-substituted proteins. These results provide direct chemical evidence that helix-2 of subunit c and helix-4 of subunit a pack close enough to each other in the membrane to interact during function. The proximity of helices supports the possibility of an interaction between Arg210 in helix-4 of subunit a and Asp61 in helix-2 of subunit c during proton translocation, as has been suggested previously.

Terpenoid-based defenses in conifers: cDNA cloning, characterization, and functional expression of wound-inducible (E)-α-bisabolene synthase from grand fir (Abies grandis)

(E)-α-Bisabolene synthase is one of two wound-inducible sesquiterpene synthases of grand fir (Abies grandis), and the olefin product of this cyclization reaction is considered to be the precursor in Abies species of todomatuic acid, juvabione, and related insect juvenile hormone mimics. A cDNA encoding (E)-α-bisabolene synthase was isolated from a wound-induced grand fir stem library by a PCR-based strategy and was functionally expressed in Escherichia coli and shown to produce (E)-α-bisabolene as the sole product from farnesyl diphosphate. The expressed synthase has a deduced size of 93.8 kDa and a pI of 5.03, exhibits other properties typical of sesquiterpene synthases, and resembles in sequence other terpenoid synthases with the exception of a large amino-terminal insertion corresponding to Pro81–Val296. Biosynthetically prepared (E)-α-[3H]bisabolene was converted to todomatuic acid in induced grand fir cells, and the time course of appearance of bisabolene synthase mRNA was shown by Northern hybridization to lag behind that of mRNAs responsible for production of induced oleoresin monoterpenes. These results suggest that induced (E)-α-bisabolene biosynthesis constitutes part of a defense response targeted to insect herbivores, and possibly fungal pathogens, that is distinct from induced oleoresin monoterpene production.