Purification and characterization of Rhodobacter sphaeroides acyl carrier protein

Cooper, Cynthia L.; Boyce, Stephen G.; Lueking, Donald R.

doi:10.1021/bi00384a013

Cited by 27 publications

(13 citation statements)

References 47 publications

(46 reference statements)

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“…The result of the gel filtration column analysis was consistent with the previously reported anomalous behavior of apo-ACP on gel filtration columns (39,40).…”

Section: Identification and Organization Of The Acps And Acppsupporting

confidence: 91%

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Biochemical and Molecular Analyses of the Streptococcus pneumoniae Acyl Carrier Protein Synthase, an Enzyme Essential for Fatty Acid Biosynthesis

McAllister

Peery

Meier

et al. 2000

Journal of Biological Chemistry

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Acyl carrier protein synthase (AcpS) is an essential enzyme in the biosynthesis of fatty acids in all bacteria. AcpS catalyzes the transfer of 4-phosphopantetheine from coenzyme A (CoA) to apo-ACP, thus converting apo-ACP to holo-ACP that serves as an acyl carrier for the biosynthesis of fatty acids and lipids. To further understand the physiological role of AcpS, we identified, cloned, and expressed the acpS and acpP genes of Streptococcus pneumoniae and purified both products to homogeneity. Both acpS and acpP form operons with the genes whose functions are required for other cellular metabolism. The acpS gene complements an Escherichia coli mutant defective in the production of AcpS and appears to be essential for the growth of S. pneumoniae. Gel filtration and cross-linking analyses establish that purified AcpS exists as a homotrimer. AcpS activity was significantly stimulated by apo-ACP at concentrations over 10 M and slightly inhibited at concentrations of 5-10 M. Double reciprocal analysis of initial velocities of AcpS at various concentrations of CoA or apo-ACP indicated a random or compulsory ordered bi bi type of reaction mechanism. Further analysis of the inhibition kinetics of the product (3,5-ADP) suggested that it is competitive with respect to CoA but mixed (competitive and noncompetitive) with respect to apo-ACP. Finally, apo-ACP bound tightly to AcpS in the absence of CoA, but CoA failed to do so in the absence of apo-ACP. Together, these results suggest that AcpS may be allosterically regulated by apo-ACP and probably proceeds by an ordered reaction mechanism with the first formation of the AcpS-apo-ACP complex and the subsequent transfer of 4-phosphopantetheine to the apo-ACP of the complex.

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“…The result of the gel filtration column analysis was consistent with the previously reported anomalous behavior of apo-ACP on gel filtration columns (39,40).…”

Section: Identification and Organization Of The Acps And Acppsupporting

confidence: 91%

“…2A). Apo-ACP has been shown to behave abnormally on gel filtration columns due to its molecular asymmetry in shape (39,40). To examine further whether apo-ACP is a dimer, purified apo-ACP was subjected to cross-linking followed by SDS-PAGE analysis.…”

Section: Identification and Organization Of The Acps And Acppmentioning

confidence: 99%

Biochemical and Molecular Analyses of the Streptococcus pneumoniae Acyl Carrier Protein Synthase, an Enzyme Essential for Fatty Acid Biosynthesis

McAllister

Peery

Meier

et al. 2000

Journal of Biological Chemistry

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“…The eighteenth amino acid, a leucine positioned four residues before the serine, was replaced by an isoleucine. Although this is a conservative substitution, it is noteworthy that the consensus leucine prevails in all other known plant ACPs and even in bacterial [7,45] and mitochondrial [5] ACPs. In other deviations from consensus, methionine substituted for threonine at position 6, while valine 56 replaced an isoleucine.…”

Section: Figmentioning

confidence: 99%

A root acyl carrier protein-II from spinach is also expressed in leaves and seeds

Schmid

Ohlrogge

1990

Plant Mol Biol

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During the synthesis of fatty acids and their utilization in plastids, fatty acyl moieties are linked to acyl carrier protein (ACP). In contrast to previously cloned organ-specific ACP isoforms, we have now isolated a cDNA clone for a potentially constitutive ACP isoform from a spinach root library. Identity between the amino acid sequence encoded by this cDNA and N-terminal sequence data for ACP-II protein from spinach leaf indicates that the root cDNA encodes ACP-II. The deduced amino acid sequence for ACP-II shows 62% identity with spinach leaf ACP-I. Southern analysis suggests that multiple ACP genes or pseudogenes occur in the spinach genome. High-stringency northern blot analysis and RNase protection studies confirm that, within the region encoding the mature ACP-II, the cloned ACP sequence is expressed in leaves and seeds as well as in roots. Quantitative RNase protection data indicate that the ratio of ACP-I and ACP-II mRNA sequences in leaf is similar to the ratio of the two proteins.

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“…6 the amino acid sequences immediately surrounding the active serine residue of acyl camer protein from mammalian, fungal, plant and bacterial FAS are shown. Similarly, the bacterial, acyl camer proteins, E.coli32 and Rhodobacter sphaeroides 33 are more homologous with each other than with any of the remaining ACP active sites.This situation also obtains for the acyl carrier protein of the fungi Saccharomyces cerevisiae and Penicillium patulum". The six sequences underline the evolutionary relationships -the rat and rabbit acyl camer proteins 31 are virtually identical.…”

Section: (C) Relationshz$ Of Fas To Other Speciesmentioning

confidence: 99%

Functional Analysis of the Fatty Acid Synthase Gene from Rattus norvegicus

1988

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meter. In diesem Fall sind diese Daten als Aki,)-Werte angegeben, also als Differenz der individuellen &(,,-Werte fiir die Paare verschiedener Verbindungen in einer binaren mobilen Phase. AuBerdem zitiert diese Publikation die theoretisch vorausberechneten AR,.,,(,]-Werte, die auf Grund der Voraussagemethode von R6qdo erhalten wurden. Magnesiumsilikat diente hier ebenfalls als stationiire Phase und eine Mischung von Methanol + Benzol als mobile Phase. Die entsprechenden Daten sind in Tab. 3 angegeben.Die Zahlwerte derTab. 3 zeigen, daB die einfache Methode der Voraussage der Retentionsparameter bei der Flussigfest-Adsorptionschromatographie, die in der vorliegenden Arbeit vorgeschlagen wurde, vollkommen vergleichbar ist mit der theoretischen Methode nach Roiy-lo. Die beobachteten Unterschiede zwischen den experimentellen und den berechneten Zahlwerten sind durch die grobe Anniiherung des Assoziationsgrades PSI in den Gln. (6a) und (6b) erklkbar. Aus diesem Grunde konnen Selbstassoziation sowie die anderen Formen intermolekularer Wechselwirkungen zwischen den Komponenten der mobilen Mischphasen noch besser fur eine theoretische Voraussage der chromatographischen Retentionsparameter ausgenutzt werden. L i t e r a t u r I L.

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Purification and characterization of Rhodobacter sphaeroides acyl carrier protein

Cited by 27 publications

References 47 publications

Biochemical and Molecular Analyses of the Streptococcus pneumoniae Acyl Carrier Protein Synthase, an Enzyme Essential for Fatty Acid Biosynthesis

Biochemical and Molecular Analyses of the Streptococcus pneumoniae Acyl Carrier Protein Synthase, an Enzyme Essential for Fatty Acid Biosynthesis

A root acyl carrier protein-II from spinach is also expressed in leaves and seeds

Functional Analysis of the Fatty Acid Synthase Gene from Rattus norvegicus

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