Cloning, Expression, and Characterization of a Human 4′-Phosphopantetheinyl Transferase with Broad Substrate Specificity

Joshi, Anil K.; Zhang, Lei; Rangan, Vangipuram S.; Smith, Stuart

doi:10.1074/jbc.m305459200

Cited by 90 publications

(108 citation statements)

References 30 publications

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“…Moreover, A. fumigatus 4'-PPTase exhibits 25 % identity (37 % similarity) to the human 4'-PPTase (Genbank accession number BC015470) and shares some significant regions of homology (WxLKExxxK) as previously noted. [9] In most organisms there is an individual 4'-PPTase for each function, however the possible presence of only one 4'-PPTase in A. fumigatus is not unique, since recent work has identified and characterised a human 4'-PPTase that appears to exhibit a broad specificity for all 4'-phosphopantetheinylation reactions including human (apo-ACP domain for cytosolic fatty acid synthetase (FAS), mitochondrial ACP, a-aminoadipate semialdehyde dehydrogenase activation (lysine catabolism)) and nonhuman (B. subtilis ACP-A (involved in fatty acid synthesis) and B. brevis tyrocidine synthetase) apo-enzymes [19,20] A new type of 4'-PPTase was also identified in Pseudomonas aeruginosa that showed association with fatty acid synthesis and siderophore metabolism. [19] Northern analysis of A. fumigatus 4'-PPTase expression indicates that the gene is constitutively expressed; this strongly suggests that protein 4'-phosphopantetheinylation is required for both primary-and secondary-metabolite production in A. fumigatus.…”

Section: Discussionmentioning

confidence: 99%

“…[20,22] In these reports, the substrate proteins exhibited molecular masses in the order of 11-38 kDa; however, in the case of baculovirus-expressed Afpes1 TCA (120 kDa), there was concern that protein mass spectrometry would not exhibit the required resolution to confirm 4'-phosphopantetheinylation. Consequently, the post-phosphopantetheinylation reaction mixture was enzymatically digested (with trypsin and V8 protease) prior to peptide mass fingerprinting.…”

Section: Discussionmentioning

confidence: 99%

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A 4′‐Phosphopantetheinyl Transferase Mediates Non‐Ribosomal Peptide Synthetase Activation in Aspergillus fumigatus

et al. 2005

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Aspergillus fumigatus is a significant human pathogen. Non‐ribosomal peptide (NRP) synthesis is thought to be responsible for a significant proportion of toxin and siderophore production in the organism. Furthermore, it has been shown that 4′‐phosphopantetheinylation is required for the activation of key enzymes involved in non‐ribosomal peptide synthesis in other species. Here we report the cloning, recombinant expression and functional characterisation of a 4′‐phosphopantetheinyl transferase from A. fumigatus and the identification of an atypical NRP synthetase (Afpes1), spanning 14.3 kb. Phylogenetic analysis has shown that the NRP synthetase exhibits greatest identity to NRP synthetases from Metarhizium anisolpiae (PesA) and Alternaria brassicae (AbrePsy1). Northern hybridisation and RT‐PCR analysis have confirmed that both genes are expressed in A. fumigatus. A 120 kDa fragment of the A. fumigatus NRP synthetase, containing a putative thiolation domain, was cloned and expressed in the baculovirus expression system. Detection of a 4′‐phosphopantetheinylated peptide (SFSAMK) from this protein, by MALDI‐TOF mass spectrometric analysis after coincubation of the 4′‐phosphopantetheinyl transferase with the recombinant NRP synthetase fragment and acetyl CoA, confirms that it is competent to play a role in NRP synthetase activation in A. fumigatus. The 4′‐phosphopantetheinyl transferase also activates, by 4′‐phosphopantetheinylation, recombinant α‐aminoadipate reductase (Lys2p) from Candida albicans, a key enzyme involved in lysine biosynthesis.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A 4′‐Phosphopantetheinyl Transferase Mediates Non‐Ribosomal Peptide Synthetase Activation in Aspergillus fumigatus

et al. 2005

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“…Acyl carrier protein is a key component of type II fatty acid synthase, which is present in the matrix (Ref. 39 and references therein) and whose role is to provide octanoate, the precursor of lipoic acid (another basic cofactor of aerobic metabolism) and long chain fatty acids that play an essential role in mitochondrial function. Because SLC25A42 functions exclusively by a counter-exchange mechanism, the carrier-mediated uptake of CoA requires efflux of a counter-substrate.…”

Section: Discussionmentioning

confidence: 99%

A Novel Member of Solute Carrier Family 25 (SLC25A42) Is a Transporter of Coenzyme A and Adenosine 3′,5′-Diphosphate in Human Mitochondria

Fiermonte

Paradies

Todisco

et al. 2009

Journal of Biological Chemistry

139

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Mitochondrial carriers are a family of proteins that transport metabolites, nucleotides, and cofactors across the inner mitochondrial membrane thereby connecting cytosolic and matrix functions. The essential cofactor coenzyme A (CoA) is synthesized outside the mitochondrial matrix and therefore must be transported into mitochondria where it is required for a number of fundamental processes. In this work we have functionally identified and characterized SLC25A42, a novel human member of the mitochondrial carrier family. The SLC25A42 gene (Haitina, T., Lindblom, J., Renström, T., and Fredriksson, R., 2006, Genomics 88, 779 -790) was overexpressed in Escherichia coli, purified, and reconstituted into phospholipid vesicles. Its transport properties, kinetic parameters, and targeting to mitochondria demonstrate that SLC25A42 protein is a mitochondrial transporter for CoA and adenosine 3,5-diphosphate. SLC25A42 catalyzed only a counter-exchange transport, exhibited a high transport affinity for CoA, dephospho-CoA, ADP, and adenosine 3,5-diphosphate, was saturable and inhibited by bongkrekic acid and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A42 is to import CoA into mitochondria in exchange for intramitochondrial (deoxy)adenine nucleotides and adenosine 3,5-diphosphate. This is the first time that a mitochondrial carrier for CoA and adenosine 3,5-diphosphate has been characterized biochemically.The mitochondrial carrier family, or the solute carrier family 25 (SLC25), 3 comprises a large group of proteins that transport a variety of substrates across the inner mitochondrial membrane and, in a few cases, across other membranes (1, 2). Common structural features of the mitochondrial carrier family members consist in a tripartite structure (three repeats of ϳ100 amino acids), the presence of two transmembrane ␣-helices separated by hydrophilic loops in each repeat, and the presence of a signature motif at the C terminus of the first helix in each repeat (Ref. 3 and references therein). The SLC25 family is by far the largest of the currently known 43 SLC families. The Saccharomyces cerevisiae genome contains 35 members, that of Arabidopsis thaliana 58, and the human genome at least 48 SLC25 members. Until now, nearly 30 members and isoforms of this family have been identified in humans. These include the uncoupling protein and the carriers for ADP/ATP, phosphate, 2-oxoglutarate/malate, citrate, carnitine/acylcarnitine, dicarboxylates, ornithine and other basic amino acids, oxodicarboxylates, deoxynucleotides and thiamine pyrophosphate, aspartate-glutamate, glutamate, S-adenosylmethionine, ATPMg/Pi, pyrimidine nucleotides, and adenine nucleotides in peroxisomes (see Ref. 1 for a review and Refs. 4 -8). The present investigation was undertaken to identify the function of SLC25A42, a novel member of the SLC25 family recently found in the human genome (9). SLC25A42 is 318 amino acids long and is highly expressed in virtually all tissues, in most at higher levels than ma...

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“…Thus, acyl-CoAs containing 2-16 C atoms were all efficiently converted to the corresponding acyl-ACPs by this enzyme. The human phosphopantetheinyl transferase has a remarkably broad tolerance not only for the acyl-S-phosphopantetheinyl donor but also for the carrier protein acceptor and can phosphopantetheinylate both peptidyl and acyl carrier proteins from prokaryotes and eukaryotes (15). Thus, the enzyme is ideally suited for routine synthesis of acyl-ACPs of any acyl chain length and ACP origin.…”

Section: Identification Of the Putative Humanmentioning

confidence: 99%

“…Isolation of Apo and Holo Forms of Human Mitochondrial ACP-The C-terminally His 6 -tagged apoACP was expressed in an Sf9/baculoviral host/vector system and purified as described earlier, and the holo-form was derived from it using human phosphopantetheinyl transferase (10,15).…”

Section: ␤-Ketoacylmentioning

confidence: 99%

Cloning, Expression, and Characterization of the Human Mitochondrial β-Ketoacyl Synthase

Zhang¹,

Joshi²,

Hofmann

et al. 2005

Journal of Biological Chemistry

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A human ␤-ketoacyl synthase implicated in a mitochondrial pathway for fatty acid synthesis has been identified, cloned, expressed, and characterized. Sequence analysis indicates that the protein is more closely related to freestanding counterparts found in prokaryotes and chloroplasts than it is to the ␤-ketoacyl synthase domain of the human cytosolic fatty acid synthase. The full-length nuclear-encoded 459-residue protein includes an N-terminal sequence element of ϳ38 residues that functions as a mitochondrial targeting sequence. The enzyme can elongate acyl-chains containing 2-14 carbon atoms with malonyl moieties attached in thioester linkage to the human mitochondrial acyl carrier protein and is able to restore growth to the respiratory-deficient yeast mutant cem1 that lacks the endogenous mitochondrial ␤-ketoacyl synthase and exhibits lowered lipoic acid levels. To date, four components of a putative type II mitochondrial fatty acid synthase pathway have been identified in humans: acyl carrier protein, malonyl transferase, ␤-ketoacyl synthase, and enoyl reductase. The substrate specificity and complementation data for the ␤-ketoacyl synthase suggest that, as in plants and fungi, in humans this pathway may play an important role in the generation of octanoyl-acyl carrier protein, the lipoic acid precursor, as well as longer chain fatty acids that are required for optimal mitochondrial function.Over the last decade, it has become clear that fungal and plant mitochondria are capable of synthesizing fatty acids de novo (1-6). The enzymes involved in the pathway are freestanding, monofunctional proteins of the type II variety and are distinct from the multifunctional polypeptide type I FASs 1 present in the cytosol of fungi and animals. Thus far, the preponderance of evidence suggests that these mitochondrial FAS pathways function to produce octanoyl-ACP, the precursor of lipoic acid (4) and longer chain-length fatty acids that may be utilized for the remodeling of mitochondrial membrane phospholipids (7). The presence of a similar mitochondrial FAS system in animals has been suspected for some time, based initially on the discovery of an ACP-like protein in animal mitochondria (8, 9). However, only recently have other components of a putative mitochondrial FAS been identified, cloned, and characterized; they include the human ACP and malonyl transferase (10) and enoyl reductase (11). In continuation of the search for other components, we have now identified and characterized a single candidate for a type II human mitochondrial ␤-ketoacyl synthase, the critical enzyme required for catalysis of the chain-elongating condensation reaction. EXPERIMENTAL PROCEDURESCloning of the Human Mitochondrial ␤-Ketoacyl Synthase-Using the sequences of several authentic type I and type II ␤-ketoacyl synthases as probes to BLAST search the human genome sequence data base, we identified a sequence (accession NP_060367) that also was represented in a human EST clone (IMAGE clone 3446492, ATCC MGC-4956) and appeared likely to encode...

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Cloning, Expression, and Characterization of a Human 4′-Phosphopantetheinyl Transferase with Broad Substrate Specificity

Cited by 90 publications

References 30 publications

A 4′‐Phosphopantetheinyl Transferase Mediates Non‐Ribosomal Peptide Synthetase Activation in Aspergillus fumigatus

A 4′‐Phosphopantetheinyl Transferase Mediates Non‐Ribosomal Peptide Synthetase Activation in Aspergillus fumigatus

A Novel Member of Solute Carrier Family 25 (SLC25A42) Is a Transporter of Coenzyme A and Adenosine 3′,5′-Diphosphate in Human Mitochondria

Cloning, Expression, and Characterization of the Human Mitochondrial β-Ketoacyl Synthase

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