Acyl carrier protein from Escherichia coli I. Aspects of the solution structure as evidenced by proton nuclear Overhauser experiments at 500 MHz

Mayo, Kevin H.; Tyrell, P. M.; Prestegard, James H.

doi:10.1021/bi00288a021

Cited by 14 publications

(18 citation statements)

References 26 publications

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“…The sequence- Leu-Ile-Met-Ala-Leu-GIu-GIu-GIu-Phe-Asp-Thr-GIu-Ile-Pro- . These data give the exact primary amino acid sequence and molecular weight (8,860) of E. coli K-12 ACP and will permit the precise interpretation of high-resolution nuclear magnetic resonance spectra (15,16,25) and X-ray crystallography patterns (17).…”

Section: Resultsmentioning

confidence: 99%

“…ACP eluted at 0.37 M NaCl, and the peak fractions were pooled, dialyzed, and lyophilized. The ACP preparations were homogeneous as judged by uv spectroscopy (21), nuclear magnetic resonance spectroscopy (15,16,25), and conformationally sensitive gel electrophoresis (11). The primary amino acid sequences of ACP and F-ACP were determined by using an Applied Biosystems gas-phase sequenator.…”

Section: Methodsmentioning

confidence: 99%

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Altered molecular form of acyl carrier protein associated with beta-ketoacyl-acyl carrier protein synthase II (fabF) mutants

Jackowski¹,

Rock²

1987

J Bacteriol

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Acyl carrier protein (ACP) is a required cofactor for fatty acid synthesis in Escherichia coli. Mutants lacking I-ketoacyl-ACP synthase II activity (fabFI orfabF3) possessed a different molecular species of ACP (F-ACP) that was separated from the normal form of the protein by conformationally sensitive gel electrophoresis. Synthase I mutants contained the normal protein. Complementation of fabFI mutants with an F' factor harboring the wild-type synthase II allele resulted in the appearance of normal ACP, whereas complementation with an F' possessing the fabF2 allele (a mutation that produces a synthase II enzyme with altered catalytic activity) resulted in the production of both forms of ACP. The structural difference between F-ACP and ACP persisted after the removal of the 4'-phosphopantetheine prosthetic group, and both forms of the protein had identical properties in an in vitro fatty acid synthase assay. Both ACP and F-ACP were purified to homogeneity, and their primary amino acid sequences were determined. The two ACP species were identical but differed from the sequence reported for E. coli E-15 ACP in that an Asn instead of an Asp was at position 24 and an Ile instead of a Val was at position 43. Therefore, F-ACP appears to be a modification of ACP that is detected when 0--ketoacyl-ACP synthase II activity is impaired.Acyl carrier protein (ACP) is one of the most abundant proteins in Escherichia coli and functions as an acyl-group carrier in bacterial fatty acid biosynthesis and as an acyl donor in membrane phospholipid biogenesis (for reviews, see references 19 and 24). The complete primary sequence of E. coli ACP is known (29). The protein contains a preponderance of acidic residues throughout the sequence and relatively few positively charged residues, which are clustered at the amino terminus. ACP has a molecular weight of 8,847, and the acyl moiety is bound as a thioester to the sulfhydryl group located at the terminus of the 4'-phosphopantetheine prosthetic group, which is attached to Ser-36 of the protein via a phosphodiester linkage. The ACP prosthetic group turnover cycle is the only known posttranslational modification of ACP.[ACP]synthase catalyzes the transfer of 4'-phosphopantetheine from coenzyme A (CoA) to apo-ACP (7), and [ACP]phosphodiesterase (28) cleaves the prosthetic group from ACP. The intracellular concentration of apo-ACP is very low (11), and the rate of prosthetic group turnover appears to be related to the cellular CoA content (12). E. coli possess two forms of P-ketoacyl-ACP synthase that catalyze the condensation of malonyl-ACP with the growing fatty acid chain (4, 9). Synthases I and II differ in their elutions from hydroxylapatite, their pH optima, their heat stabilities, and their substrate specificities and are encoded by distinct structural genes (for reviews, see references 5 and 23). Synthase I mutants (fabB) are unable to synthesize unsaturated fatty acids and require an exogenous unsaturated fatty acid supplement for growth (26). Strains that lack 3-ketoacyl-ACP sy...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Altered molecular form of acyl carrier protein associated with beta-ketoacyl-acyl carrier protein synthase II (fabF) mutants

Jackowski¹,

Rock²

1987

J Bacteriol

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“…6 D and E) the large absorptive resonance near 3.0 ppin is due to a nuclear Overhauser effect at the Tyr71 CPH [22] following ring proton polarization. The magnitude of this effect is the result of several factors which include the weighted average distance between the ring protons and CPH, their relative motions, the spectrometer frequency, etc.…”

Section: Photo-cidnp ' H Nmrmentioning

confidence: 95%

Divalent cation biding to reduced and octanoyl acyl‐carrier protein

Tener

Mayo

1990

European Journal of Biochemistry

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Mn(I1) EPR binding studies with reduced acyl-carrier protein (ACP-SH) strongly suggest the presence of two relatively high-affinity manganese-binding sites (average &/site z 80 pM) at physiological pH. Lowering the pH or titrating with sodium chloride reduces the average number of bound divalent cations and decreases the binding affinity. This is consistent with the idea that anionic ligand(s), e. g. the carboxylate of glutamic or aspartic acid, on the protein are involved in manganese ion coordination. At pH values above 8.0, binding affinity is also reduced, whereas the average number of bound metal ions increases to about five at pH 8.5. By interacting weakly with divalent cations (average &/site z 1 mM), octanoyl acyl-carrier protein (OcoACP) exhibits dramatically different metal-ion-binding properties compared to ACP-SH. Calcium and magnesium can compete in either ACP species for manganese binding. Photochemically-induced dynamic nuclear polarisation 'H-NMR experiments strongly suggest that ACP-SH and OcoACP undergo at pH-induced conformational change between pH 5.5 and pH 7.0, and that divalent cations stabilize the protein against such pH-induced structural perturbations.Fatty acid biosynthesis is a central biochemical pathway involving several enzymatic steps [I -31. Acyl-carrier proteins (ACP) are coenzymes which function in acyl-group-transfer reactions, chain-elongation reactions by successive addition of two-carbon fragments from malonyl-CoA, and as anchors for reduction and dehydration reactions that ultimately lead to a saturated fatty acid [2]. Since ACP is active in a variety of differing acylated states involved in various enzymatic reactions and, therefore, with a number of different enzymes, it is likely that ACP has the ability to modify or fine-tune its conformation to interact efficiently in all situations and, perhaps, even effect regulation of fatty acid biosynthesis.The ability to isolate ACP from Escherichiu coli as a soluble protein, and not as part of a tightly associated protein complex found in higher organisms, has made it the most studied among all ACPs which, in any event, possess a high degree of sequence similarity [2]. Of the 77 amino acids (8847 Da) found in E. coli ACP, 22 are acidic (glutamic and aspartic acid residues) and six are basic (lysine, arginine and histidine residues) [4, 51. This gives the protein an net charge of -16. To date, a secondary backbone structure for E. coli ACP-SH (reduced) has been derived from two-dimensional 'H-NMR studies [6]. The following segments are proposed to be helical: residues 3 -15,37 -51,56 -63 and 65 -75; these are connected by extended chain and p-turn structure. The three-dimensional structure of ACP-SH, determined by NMR pseudo-energy and distance geometry calculations [7], have identified three helical regions: residues 3-14, 37-51 and 65-75, which run anti-parallel to each other. Octanoyl-ACP, an S-acylated species of ACP, shows a similar overall conformation with Correspondence to K .

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“…We have reported over the last few years several stages in the development of a structure from one-and twodimensional NMR work. This has included assignment of resonances in the proton NMR spectrum (Mayo et al, 1983; Holak & Prestegard, 1986), determination of secondary structure (Holak & Prestegard, 1986), and determination of three-dimensional characteristics of several short segments (Holak et al, 1987a). We present here a complete structure for the protein and a preliminary discussion of chemical aspects of potential importance in acyl chain binding.…”

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confidence: 99%

Three-dimensional structure of acyl carrier protein determined by NMR pseudoenergy and distance geometry calculations

Holak

Kearsley²,

Kim³

et al. 1988

Biochemistry

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108

100

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Distance constraints from two-dimensional NMR cross-relaxation data are used to derive a three-dimensional structure for acyl carrier protein from Escherichia coli. Several approaches to structure determination are explored. The most successful proves to be an approach that combines the early stages of a distance geometry program with energy minimization in the presence of NMR constraints represented as pseudopotentials. Approximately 450 proton to proton distance constraints including 50 long-range constraints were included in these programs. Starting structures were generated at random by the distance geometry program and energies minimized by a molecular mechanics module to give final structures. Seven of the structures were deemed acceptable on the basis of agreement with experimentally determined distances. Root-mean-square deviations from the mean of these structures for backbone atoms range from 2 to 3 A. All structures show three roughly parallel helices with hydrophobic residues facing inward and hydrophilic residues facing outward. A hydrophobic cleft is recognizable and is identified as a likely site for acyl chain binding.

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Acyl carrier protein from Escherichia coli I. Aspects of the solution structure as evidenced by proton nuclear Overhauser experiments at 500 MHz

Cited by 14 publications

References 26 publications

Altered molecular form of acyl carrier protein associated with beta-ketoacyl-acyl carrier protein synthase II (fabF) mutants

Altered molecular form of acyl carrier protein associated with beta-ketoacyl-acyl carrier protein synthase II (fabF) mutants

Divalent cation biding to reduced and octanoyl acyl‐carrier protein

Three-dimensional structure of acyl carrier protein determined by NMR pseudoenergy and distance geometry calculations

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