Use of 13C in biosynthetic studies. The labeling pattern in dihydrofusarubin enriched from [13C]- and [13C, 2H]acetate in cultures of Fusarium solani

1980 ◽  
Vol 58 (13) ◽  
pp. 1380-1385 ◽  
Author(s):  
Itsuo Kurobane ◽  
Leo C. Vining ◽  
A. Gavin McInnes ◽  
John A. Walter
Keyword(s):  
Fusarium Solani ◽  
Carrier Protein ◽  
Single Chain ◽  
Acetyl Coa ◽  
Biotin Carboxyl Carrier Protein ◽  
H Nmr ◽  
Malonyl Coa ◽  
Starter Unit ◽  
Biosynthetic Studies ◽  
Labeling Pattern

The pattern of 13C and 2H incorporation from [1-13C]-, [2-13C]-, [1-13C0;1;1,2-13C1;0;1]-, and [2-13C0;1,2-2H3;3]acetate into dihydrofusarubin 1, produced by cultures of Fusariumsolani, has been determined by 13C and 2H nmr of the derivatives anhydrofusarubin 3 and anhydrofusarubin diacetate 4. The results show that 1 is biosynthesized from seven uniformly-incorporated acetate units with C-3, C-11 originating from the "starter" unit. They strongly suggest that linear head-to-tail condensation of an acetate and six malonate units forms a single-chain heptaketide intermediate. The evidence also suggests that, during conversion of [13C, 2H]-labeled acetyl-CoA to malonyl-CoA, 2H is transferred to biotin carboxyl carrier protein where it does not exchange rapidly with the medium and is available for conversion of endogenous malonyl-CoA to [2H]-enriched acetyl-CoA.

scholarly journals The Three-Dimensional Structure of the Biotin Carboxylase-Biotin Carboxyl Carrier Protein Complex of E. coli Acetyl-CoA Carboxylase

Structure ◽  
2013 ◽  
Vol 21 (4) ◽  
pp. 650-657 ◽  
Author(s):  
Tyler C. Broussard ◽  
Matthew J. Kobe ◽  
Svetlana Pakhomova ◽  
David B. Neau ◽  
Amanda E. Price ◽  
...  
Keyword(s):  
Protein Complex ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Carrier Protein ◽  
Biotin Carboxylase ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Biotin Carboxyl Carrier Protein ◽  
Three Dimensional Structure ◽  
E Coli

Biotin carboxyl carrier protein isoforms in Brassicaceae oilseeds

2000 ◽  
Vol 28 (6) ◽  
pp. 595-598 ◽  
Author(s):  
J. J. Thelen ◽  
S. Mekhedov ◽  
J. B. Ohlrogge
Keyword(s):  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Single Gene ◽  
Protein Isoforms ◽  
Carrier Protein ◽  
Biotin Carboxyl Carrier Protein ◽  
Developing Seeds ◽  
Malonyl Coa ◽  
Evolutionarily Conserved ◽  
Plant Arabidopsis Thaliana

De novo fatty acid biosynthesis occurs predominantly in plastids. The committed step for this pathway is the production of malonyl-CoA catalysed by acetyl-CoA carboxylase (ACCase). In most plants, plastidial ACCase is a multisubunit complex minimally comprised of four polypeptides, which catalyse two reactions. In the simple oilseed plant, Arabidopsis thaliana two cDNAs encoding biotin carboxyl carrier protein (BCCP) isoforms have been identified. The remaining three subunits of ACCase appear to be single gene members in A. thaliana [Mekhedov, Martinez de Ilarduya and Ohlrogge (2000) Plant Physiol. 122, 389–401]. Transcript and protein analyses indicate that BCCP isoform 1 is constitutively expressed while isoform 2 is predominantly expressed in developing seeds. The apparent masses of constitutive and seed-enriched BCCP isoforms agree with the apparent masses of recombinantly expressed isoforms 1 and 2, respectively. In a related oilseed, Brassica napus multiple putative BCCP polypeptides were also observed in developing seeds. The presence of a divergent class of BCCP genes in A. thaliana and B. napus coincident with appropriately sized biotin-containing proteins expressed specifically in developing seeds, suggests that these BCCPs play an evolutionarily conserved role in oil deposition.

scholarly journals Biotin carboxyl carrier protein and carboxyltransferase subunits of the multi-subunit form of acetyl-CoA carboxylase from Brassica napus: cloning and analysis of expression during oilseed rape embryogenesis

1996 ◽  
Vol 315 (1) ◽  
pp. 103-112 ◽  
Author(s):  
Kieran M. ELBOROUGH ◽  
Robert WINZ ◽  
Ranjit K. DEKA ◽  
Jonathan E. MARKHAM ◽  
Andrew J. WHITE ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Embryo Development ◽  
Oilseed Rape ◽  
Northern Analysis ◽  
Carrier Protein ◽  
Type I ◽  
Type Ii ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Biotin Carboxyl Carrier Protein

In the oilseed rape Brassica napus there are two forms of acetyl-CoA carboxylase (ACCase). As in other dicotyledonous plants there is a type I ACCase, the single polypeptide 220 kDa form, and a type II multi-subunit complex analogous to that of Escherichia coli and Anabaena. This paper describes the cloning and characterization of a plant biotin carboxyl carrier protein (BCCP) from the type II ACCase complex that shows 61% identity/79% similarity with Anabaena BCCP at the amino acid level. Six classes of nuclear encoded oilseed rape BCCP cDNA were cloned, two of which contained the entire coding region. The BCCP sequences allowed the assignment of function to two previously unassigned Arabidopsis expressed sequence tag (EST) sequences. We also report the cloning of a second type II ACCase component from oilseed rape, the β-carboxyltransferase subunit (βCT), which is chloroplast-encoded. Northern analysis showed that although the relative levels of BCCP and βCT mRNA differed between different oilseed rape tissues, their temporal patterns of expression were identical during embryo development. At the protein level, expression of BCCP during embryo development was studied by Western blotting, using affinity-purified anti-biotin polyclonal sera. With this technique a 35 kDa protein thought to be BCCP was shown to reside within the chloroplast. This analysis also permitted us to view the differential expression of several unidentified biotinylated proteins during embryogenesis.

scholarly journals The fractionation of the fatty acid synthetase activities of avocado mesocarp plastids

1975 ◽  
Vol 146 (2) ◽  
pp. 439-445 ◽  
Author(s):  
P J Weaire ◽  
R G O Kekwick
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
De Novo ◽  
Acyl Carrier Protein ◽  
Fatty Acid Synthetase ◽  
Carrier Protein ◽  
Acetyl Coa ◽  
Principal Product ◽  
Absolute Requirement ◽  
Malonyl Coa

1. The range of fatty acids formed by preparations of ultrasonically ruptured avocado mesocarp plastids was dependent on the substrate. Whereas [1-14C]palmitate and [14C]oleate were the major products obtained from [-14C]acetate and [1-14C]acetyl-CoA, the principal product from [2-14C]malonyl-CoA was [14-C]stearate. 2. Ultracentrifugation of the ruptured plastids at 105000g gave a supernatant that formed mainly stearate from [2-14C]malonyl-CoA and to a lesser extent from [1-14C]acetate. The incorporation of [1-14C]acetate into stearate by this fraction was inhibited by avidin. 3. The 105000g precipitate of the disrupted plastids incorporated [1-14C]acetate into a mixture of fatty acids that contained largely [14C]plamitate and [14C]oleate. The formation of [14C]palmitate and [14C]oleate by disrupted plastids was unaffected by avidin. 4. The soluble fatty acid synthetase was precipitated from the 105000g supernatant in the 35-65%-saturated-(NH4)2SO4 fraction and showed an absolute requirement for acyl-carrier protein. 5. Both fractions synthesized fatty acids de novo.

scholarly journals Characterization of a Bifunctional Archaeal Acyl Coenzyme A Carboxylase

2003 ◽  
Vol 185 (3) ◽  
pp. 938-947 ◽  
Author(s):  
Songkran Chuakrut ◽  
Hiroyuki Arai ◽  
Masaharu Ishii ◽  
Yasuo Igarashi
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Coenzyme A ◽  
Amino Acid Sequences ◽  
Carrier Protein ◽  
Subunit Structure ◽  
Biotin Carboxylase ◽  
Acetyl Coa ◽  
Biotin Carboxyl Carrier Protein ◽  
Acyl Coenzyme A

ABSTRACT Acyl coenzyme A carboxylase (acyl-CoA carboxylase) was purified from Acidianus brierleyi. The purified enzyme showed a unique subunit structure (three subunits with apparent molecular masses of 62, 59, and 20 kDa) and a molecular mass of approximately 540 kDa, indicating an α4β4γ4 subunit structure. The optimum temperature for the enzyme was 60 to 70°C, and the optimum pH was around 6.4 to 6.9. Interestingly, the purified enzyme also had propionyl-CoA carboxylase activity. The apparent Km for acetyl-CoA was 0.17 ± 0.03 mM, with a V max of 43.3 ± 2.8 U mg−1, and the Km for propionyl-CoA was 0.10 ± 0.008 mM, with a V max of 40.8 ± 1.0 U mg−1. This result showed that A. brierleyi acyl-CoA carboxylase is a bifunctional enzyme in the modified 3-hydroxypropionate cycle. Both enzymatic activities were inhibited by malonyl-CoA, methymalonyl-CoA, succinyl-CoA, or CoA but not by palmitoyl-CoA. The gene encoding acyl-CoA carboxylase was cloned and characterized. Homology searches of the deduced amino acid sequences of the 62-, 59-, and 20-kDa subunits indicated the presence of functional domains for carboxyltransferase, biotin carboxylase, and biotin carboxyl carrier protein, respectively. Amino acid sequence alignment of acetyl-CoA carboxylases revealed that archaeal acyl-CoA carboxylases are closer to those of Bacteria than to those of Eucarya. The substrate-binding motifs of the enzymes are highly conserved among the three domains. The ATP-binding residues were found in the biotin carboxylase subunit, whereas the conserved biotin-binding site was located on the biotin carboxyl carrier protein. The acyl-CoA-binding site and the carboxybiotin-binding site were found in the carboxyltransferase subunit.

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