Purification and NMR characterization of acyl carrier protein

The acyl carrier protein preparation obtained using the 2-propanol method of Rock and Cronan (Rock, C. O., and Cronan, J. E., Jr. (1981) Methods Enzymol. 71, 341-351) can be further purified with reversed-phase high-performance liquid chromatography. A homogeneous sample of acyl carrier protein is obtained as determined by NMR and reversed-phase high-performance liquid chromatography.     

Purification and characterization of acyl carrier protein from two cyanobacteria species

The acyl carrier protein (ACP), an essential protein cofactor for fatty acid synthesis, has been isolated from two cyanobacteria: the filamentous, heterocystous, Anabaena variabilis (ATCC 29211) and the unicellular Synechocystis 6803 (ATCC 27184). Both ACPs have been purified to homogeneity utilizing a three-column procedure. Synechocystis 6803 ACP was purified 1800-fold with 67% yield, while A. variabilis ACP was purified 1040-fold with 50% yield. Yields of 13.0 micrograms ACP/g Synechocystis 6803 and 9.0 micrograms ACP/g A. variabilis were achieved. Amino acid analysis indicated that these ACPs were highly charged acidic proteins similar to other known ACPs. Sequence analysis revealed that both cyanobacterial ACPs were highly conserved with both spinach and Escherichia coli ACP at the phosphopantetheine prosthetic group region. Examining the probability of alpha-helix and beta-turn regions in various ACPs, showed that cyanobacterial ACPs were more closely related to E. coli ACP than spinach ACP I. Immunoblot analysis and a competitive binding assay for ACP illustrated that both ACPs bound poorly to spinach ACP I antibody. SDS/PAGE and native PAGE of Synechocystis 6803 ACP and A. variabilis ACP showed that cyanobacteria ACPs co-migrated with E. coli ACP and had relative molecular masses of 18,100 and 17,900 respectively. Both native and urea gel analysis of acyl-ACP products from fatty acid synthase reactions demonstrated that bacterial ACPs and plant ACP gave essentially the same metabolic products when assayed using either bacterial or plant fatty acid synthase. A. variabilis and Synechocystis 6803 ACP could be acylated using E. coli acyl ACP synthetase.     

Purification and characterization of the flagellar basal body of Rhodobacter sphaeroides

Flagellar hook-basal body (HBB) complexes were purified from Rhodobacter sphaeroides. The HBB was more acid labile but more heat stable than that of Salmonella species, and protein identification revealed that HBB components were expressed only from one of the two sets of flagellar gene clusters on the R. sphaeroides genome, under the heterotrophic growth conditions tested here.     

Purification and characterization of recombinant spinach acyl carrier protein I expressed in Escherichia coli

Expression of plant acyl carrier protein (ACP) in Escherichia coli at levels above that of constitutive E. coli ACP does not appear to substantially alter bacterial growth or fatty acid metabolism. The plant ACP expressed in E. coli contains pantetheine and approximately 50% is present in vivo as acyl-ACP. We have purified and characterized the recombinant spinach ACP-I. NH2-terminal amino acid sequencing indicated identity to authentic spinach ACP-I, and there was no evidence for terminal methionine or formylmethionine. Recombinant ACP-I was found to completely cross-react immunologically with polyclonal antibody raised to spinach ACP-I. Recombinant ACP-I was a poor substrate for E. coli fatty acid synthesis. In contrast, Brassica napus fatty acid synthetase gave similar reaction rates with both recombinant and E. coli ACP. Similarly, malonyl-coenzyme A:acyl carrier protein transacylase isolated from E. coli was only poorly able to utilize the recombinant ACP-I while the same enzyme from B. napus reacted equally well with either E. coli ACP or recombinant ACP-I. E. coli acyl-ACP synthetase showed a higher reaction rate for recombinant ACP-I than for E. coli ACP. Expression of spinach ACP-I in E. coli provides, for the first time, plant ACP in large quantities and should aid in both structural analysis of this protein and in investigations of the many ACP-dependent reactions of plant lipid metabolism.     

Purification and partial characterization of acyl carrier protein from Euglena gracilis variety bacillaris.

Acyl carrier protein (ACP) was purified from Euglena gracilis variety bacillaris in yields of about 1 mg/100 g (wet wt) of cells. Antibodies against the purified protein were raised in hens and isolated from eggs. Antibodies raised against Euglena ACP inhibited the Euglena chloroplast nonaggregated fatty acid synthetase using either Euglena or Escherichia coli ACP as a substrate. Comparisons with other ACPs included the following items: biologic activity, acidic pI, size, behavior in size exclusion media, and amino acid sequence of the N-terminal portion of the molecule.     

Purification and characterization of 3,4-dihydroxyphenylalanine oxidative deaminase from Rhodobacter sphaeroides OU5

An enzyme involved in the catabolism of 3,4-dihydroxyphenylalanine (DOPA) was isolated from Rhodobacter sphaeroides OU5. The enzyme catalyzes the formation of 3,4-dihydroxyphenylpyruvic acid (DOPP) and ammonia from DOPA. Formation of ammonia by DOPA oxidative deaminase was O2 dependent and the enzyme isolated to its homogeneity has 100% affinity for DOPA. DOPA oxidative deaminase is functional at low concentrations of the substrate (< 100 micromol.L(-1)) and is independent of NADH. The molecular mass of the purified enzyme is approximately 190 kDa and the enzyme could be a pentamer of 54, 42, 34, 25, and 23 kDa subunits as determined by SDS-PAGE.     

Characterization of a novel enoyl-acyl carrier protein reductase of diazaborine-resistant Rhodobacter sphaeroides mutant

Rhodobacter sphaeroides contains two enoyl-acyl carrier protein (ACP) reductases, FabI(1) and FabI(2). However, FabI(1) displays most of the cellular enzyme activity. The spontaneous diazaborine-resistant mutation was mapped as substitution of glutamine for proline 155 (P155Q) of FabI(1). The mutation of FabI(1)[P155Q] increased the specificity constants (k(cat)/K(m)) for crotonyl-ACP and NADH by more than 2-fold, while the site-directed mutation G95S (FabI(1)[G95S]), corresponding to the well-known G93 mutation of Escherichia coli FabI, rather decreased the values. Inhibition kinetics of the enzymes revealed that triclosan binds to the enzyme in the presence of NAD(+), while the diazaborine appears to interact with NADH and NAD(+) in the enzyme active site. The apparent inhibition constant K(i)(') of triclosan for FabI(1)[P155Q] and FabI(1)[G95S] at saturating NAD(+) were approximately 80- and 3-fold higher than that for the wild-type enzyme, respectively, implying that the inhibition was remarkably impaired by the P155Q mutation. The similar levels of K(i)(') of diazaborine for the mutant enzymes were also observed with respect to NAD(+). Thus, the novel mutation P155Q appears to disturb the binding of inhibitors to the enzyme without affecting the catalytic efficiency.     

浑球红假单胞菌（Rhodobacter sphaeroides）谷氨酸合酶的纯化及性质

浑球红假单胞菌菌株601经超声击碎,粗提液通过Triton处理,硫酸铵沉淀,DE—52和DEAE—sephadex A—50柱层析及 Seqhadex G—200凝胶过滤等步骤,将谷氨酸合酶(GOGAT)分离纯化,在聚丙烯酰胺凝胶电泳上呈现一条带。GOGAT表观分子量约为138 kD。该酶最大光吸收在278,375,450 nm和475 nm处,表明GOGAT可能是一种黄素蛋白。纯化的GOGAT对其底物 Gln,α—酮戊二酸和NADPH的表观K_m值分别为830,150和6μmol/L。反应产物Gln和NADP,几种氨基酸对GOGAT活力有不同程度的抑制作用,Gln类似物DON对GOGAT活力有强烈的抑制作用。     

Purification and characterization of the chaperonin 10 and chaperonin 60 proteins from Rhodobacter sphaeroides.

Two heat-shock proteins that show high identity with the Escherichia coli chaperonin 60 (groEL) and chaperonin 10 (groES) chaperonin proteins were purified and characterized from photolithoautotrophically grown Rhodobacter sphaeroides. The proteins were purified by using sucrose density gradient centrifugation and Mono-Q anion-exchange chromatography. In the presence of 1 mM ATP, the chaperonin 10 and chaperonin 60 proteins bound to each other and comigrated as a large complex during sucrose density gradient centrifugation. The native molecular weights of each protein as determined by gel filtration chromatography were 889,200 for chaperonin 60 and 60,000 for chaperonin 10. Chaperonin 60 is comprised of monomers with a molecular weight of 61,000 and chaperonin 10 is comprised of monomers with a molecular weight of 12,700 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chaperonin 60 was 9.3% of the total soluble cell protein during photolithoautotrophic growth which increased to 28.5% following heat-shock treatment. When cells were grown photoheterotrophically or chemoheterotrophically, chaperonin 60 was reduced to 6.7% and 3.5%, respectively, of the total soluble protein. The N-terminal amino acid sequence of each protein was determined; chaperonin 60 of R. sphaeroides showed 72% identity to E. coli chaperonin 60 protein, and R. sphaeroides chaperonin 10 showed 45% identity with E. coli chaperonin 10. R. sphaeroides chaperonin 60 catalyzed ATP hydrolysis with a specific activity of 134 nmol min-1 mg-1 (kcat = 0.13 s-1) and was inhibited by R. sphaeroides chaperonin 10, but not E. coli chaperonin 10. The E. coli chaperonin 60 ATPase activity was inhibited by chaperonin 10 from both R. sphaeroides and E. coli.     

Purification and characterization of the acyl carrier protein of the Streptomyces glaucescens tetracenomycin C polyketide synthase.

The acyl carrier protein (ACP) of the tetracenomycin C polyketide synthase, encoded by the tcmM gene, has been expressed in both Streptomyces glaucescens and Escherichia coli and purified to homogeneity. Expression of the tcmM gene in E. coli results mainly in the TcmM apo-ACP, whereas expression in S. glaucescens yields solely the holo-ACP. The purified holo-TcmM is active in a malonyl coenzyme A:ACP transacylase assay and is labeled by radioactive beta-alanine, confirming that it carries a 4'-phosphopantetheine prosthetic group.     

Identification and characterization of a GroEL homologue in Rhodobacter sphaeroides

A protein closely related to the Escherichia coli GroEL protein has been isolated from Rhodobacter sphaeroides. Native and SDS-polyacrylamide gel electrophoresis of this protein have shown that it is present in the cell as a multimeric complex of Mr 670,000 which is composed of a monomer of Mr 58,000. Antisera raised against the Mr 58,000 polypeptide have been shown to cross-react with GroEL and the alpha subunit of the pea plastid chaperonin. The N-terminal amino acid sequence of the Mr 58,000 polypeptide is identical to that of GroEL at 15 of 19 residues and is also closely related to the alpha subunit of the pea plastid chaperonin, though less so to the beta subunit.     

Requirements for chemotaxis protein localization in Rhodobacter sphaeroides

Many proteins have recently been shown to localize to different regions of the bacterial cell. This is most striking in the case of the Escherichia coli chemotaxis pathway in which the components localize at the cell poles. Rhodobacter sphaeroides has a more complex chemotaxis system with two complete pathways, each localizing to different positions, one pathway at the pole and one at a discrete cluster within the cytoplasm of the bacterium. Using genomic replacement of the wild-type chemotaxis genes in R. sphaeroides with their corresponding fluorescent protein fusions in conjunction with in frame deletions of other chemotaxis genes, we have investigated which proteins are required for the formation of the polar and cytoplasmic chemotaxis protein clusters. As in E. coli, the polarly targeted CheA and CheW homologues are required for the formation of the polar cluster. However, the formation of the cytoplasmic cluster requires the cytoplasmic chemoreceptors and CheW but not the CheAs. Interestingly, even when deletion of a component resulted in the chemotaxis proteins of one pathway becoming delocalized and diffuse in the cytoplasm, in no case were any chemotaxis proteins seen to localize to the other signalling cluster.     

Isolation and characterization of a virulent phage for Rhodobacter sphaeroides

A new virulent bacteriophage, termed øRsV, was isolated from a local sewage plant on the facultative phototrophic bacterium Rhodobacter sphaeroides DSM 159 as the host organism. Electron microscopic studies revealed that in general morphology phage øRsV resembles the T-even Escherichia coli phages. The host range of phage øRsV was restricted to strains of R. sphaeroides. E. coli strains B and K 12 were not infected. The phage genome was characterized on the basis of thermal denaturation profiles and restriction analyses indicating that it consists of about 160 kb of double-stranded DNA lacking cohesive ends. The G+C content was determined to be 46.8 mol%.     

Structural homology of spinach acyl carrier protein and Escherichia coli acyl carrier protein based on NMR data

Acyl carrier proteins (ACPs) from spinach and from Escherichia coli have been used to demonstrate the utility of proton NMR for comparison of homologous structures. The structure of E. coli ACP had been previously determined and modeled as a rapid equilibrium among multiple conformational forms (Kim and Prestegard, Biochemistry 28:8792–8797, 1989). Spinach ACP showed two slowly exchanging forms and could be manipulated into one form for structural study. Here we compare this single form to postulated multiple forms of E. coli ACP using the limited amount of NOE data available for the spinach protein. A number of long-range NOE contacts were present between homologous residues in both spinach and E. coli ACP, suggesting tertiary structural homology. To allow a more definitive structural comparison, a method was developed to use spinach ACP NOE constraints to search for regions of structural divergence from two postulated forms of E. coli ACP. The homologous regions of the two protein sequences were aligned, additional distance constraints were extracted from the E. coli structure, and these were mapped onto the spinach sequence. These distance constraints were combined with experimental NOE constraints and a distance geometry simulated annealing protocol was used to test for compatibility of the constraints. All of the experimental spinach NOE constraints could be successfully combined with the E. coli data, confirming the general hypothesis of structural homology. A better fit was obtained with one form, suggesting a preferential stabilization of that form in the spinach case. Proteins 27:131–143 © 1997 Wiley-Liss, Inc.