Molecular cloning and sequence analysis of complementary DNA encoding rat mammary gland medium-chain S-acyl fatty acid synthetase thio ester hydrolase

Safford, Richard; Silva, J. de; Lucas, C.; Windust, J.; Shedden, J.; James, Christopher M.; Sidebottom, C.; Slabas, Antoni R.; Tombs, M. P.; Hughes, Stephen

doi:10.1021/bi00379a023

Cited by 32 publications

(15 citation statements)

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“…The data base search revealed that ORF1 and ORF2, as shown previously for GRST (30), were also similar to predicted vertebrate thioesterase II enzymes from duck (DFAT [45]) and rat (RFAT [47]) which can transfer and thus catalyze release of fatty acids from multifunctional synthetase complexes (Table 2 and Fig. 8).…”

Section: And Discussionsupporting

confidence: 76%

“…2. GRST, grsT gene product (30); RFAT, rat medium-chain fatty acid thioesterase (47); DFAT, duck medium-chain fatty acid thioesterase (45); Ac Tr (yeast), yeast acetyltransferase (50); P/M Tr (yeast), yeast palmytl/malonyltransferase (50); Ac/M Tr (chicken), chicken acetyl/malonyltransferase (11); EryA(1 to 3), three thioesterase motifs predicted from the erythromycin synthetase gene sequence (12); ACVS, ACV synthetase from A. nidulans (55).…”

Section: And Discussionmentioning

confidence: 99%

“…We predict that the alanine residues in bialaphos are added [30]), DFAT (duck fatty acid thioesterase [45]), and RFAT (rat fatty acid thioesterase [47]). Shaded regions indicate positions where similar amino acids (R = K; D = E; F = Y = W; M = I = L = V) were found in three of the five proteins.…”

Section: And Discussionmentioning

confidence: 99%

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Nucleotide sequence analysis reveals linked N-acetyl hydrolase, thioesterase, transport, and regulatory genes encoded by the bialaphos biosynthetic gene cluster of Streptomyces hygroscopicus

et al. 1991

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Nucleotide sequence analysis of a 5,000-bp region of the bialaphos antibiotic production (bap) gene cluster defined five open reading frames (ORFs) which predicted structural genes in the order bah, ORF1, ORF2, and ORF3 followed by the regulatory gene, brpA (H. Anzai, T. Murakami, S. Imai, A. Satoh, K. Nagaoka, and C. J. Thompson, J. Bacteriol. 169:3482-3488, 1987). The four structural genes were translationally coupled and apparently cotranscribed from an undefined promoter(s) under the positive control of the brpA gene product. S1 mapping experiments indicated that brpA was transcribed by two promoters (brpAp1 and brpAp2) which initiate transcription 150 and 157 bp upstream of brpA within an intergenic region and at least one promoter further upstream within the bap gene cluster (brpAp3). All three transcripts were present at low levels during exponential growth and increased just before the stationary phase. The levels of the brpAp3 band continued to increase at the onset of stationary phase, whereas brpApl-and brpAp2-protected fragments showed no further change. BrpA contained a possible helix-turn-helix motif at its C terminus which was similar to the C-terminal regulatory motif found in the receiver component of a family of two-component transcriptional activator proteins. This motif was not associated with the N-terminal domain conserved in other members of the family. The structural gene cluster sequenced began with bah, encoding a bialaphos acetylhydrolase which removes the N-acetyl group from bialaphos as one of the final steps in the biosynthetic pathway. The observation that Bah was similar to a rat and to a bacterial (Acinetobacter calcoaceticus) lipase probably reflects the fact that the ester bonds of triglycerides and the amide bond linking acetate to phosphinothricin are similar and hydrolysis is catalyzed by structurally related enzymes. This was followed by two regions encoding ORF1 and ORF2 which were similar to each other (48% nucleotide identity, 31% amino acid identity), as well as to GrsT, a protein encoded by a gene located adjacent to gramicidin S synthetase in Bacillus brevis, and to vertebrate (mallard duck and rat) thioesterases. The amino acid sequence and hydrophobicity proffle of ORF3 indicated that it was related to a family of membrane transport proteins. It was strikingly similar to the citrate uptake protein encoded by the transposon Tn3411.Bialaphos, a linear tripeptide produced as a secondary metabolite by Streptomyces hygroscopicus, is composed of two L-alanine residues and the glutamate analog phosphinothricin (PT) (4, 29). After cleavage of bialaphos by intracellular nonspecific peptidases, PT acts as an inhibitor of glutamine synthetase (4, 29). For this reason, bialaphos has herbicide and antibiotic activities. The biosynthetic pathway has been well characterized by analyzing intermediates accumulated in cultures of blocked mutants (19). Primary metabolic substrates are converted to the demethylated form of PT, which is then condensed with two alanine residues. Althoug...

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Section: And Discussionsupporting

confidence: 76%

Section: And Discussionmentioning

confidence: 99%

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Nucleotide sequence analysis reveals linked N-acetyl hydrolase, thioesterase, transport, and regulatory genes encoded by the bialaphos biosynthetic gene cluster of Streptomyces hygroscopicus

et al. 1991

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“…However, in some specialized tissues, such as nonruminant mammary glands [2,3] and avian uropygial glands [4], a discrete monofunctional monomeric protein, thioesterase II, catalyses the hydrolysis of medium-chain fatty acids from the fatty acid synthase. The amino acid sequences of both the thioesterase I domain of the rat fatty acid synthase [5] and the thioesterase II enzyme from lactating rat mammary gland [6,7] have been determined, and their cDNAs have been cloned. The two types of chain-terminating enzyme share some common structural features and appear to be distantly related [5].…”

Section: Introductionmentioning

confidence: 99%

Expression in Escherichia coli, purification and characterization of two mammalian thioesterases involved in fatty acid synthesis

Naggert

Witkowski

Wessa

et al. 1991

Biochemical Journal

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Thioesterase I, a constituent domain of the multifunctional fatty acid synthase, and thioesterase II, an independent monofunctional protein, catalyse the chain-terminating reaction in fatty acid synthesis de novo at long and medium chain lengths respectively. The enzymes have been cloned and expressed in Escherichia coli under the control of the temperaturesensitive A repressor. The recombinant proteins are full-length catalytically competent thioesterases with specificities indistinguishable from those of the natural enzymes.

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“…TE II has broad substrate specificity and interacts with FAS to hydrolyze the fatty acyl thioesters before they mature to longerchain acids. The cDNA sequences of the TE Ils of the rat mammary gland (6) and of the uropygial gland of mallard ducks (7) have been reported. We recently showed by site-directed mutagenesis that the Ser10'l and His274 of TE I are important in its mechanism of action (2,3).…”

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confidence: 99%

Roles of Ser101, Asp236, and His237 in catalysis of thioesterase II and of the C-terminal region of the enzyme in its interaction with fatty acid synthase.

Tai

Chirala

Wakil

1993

Proc. Natl. Acad. Sci. U.S.A.

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Thioesterase II (TE H), present in specialzed tissues, catalyzes the chain termination and release of mediumchain fatty acids from fatty acid synthase [FAS; acylCoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl-and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85]. We have expressed rat mammary gland TE I in Escherichia coli and created several site-directed mutants. Replacing both Ser"l" and His237 with Ala yielded inactive proteins, suggetng that these residues are part of the catalytic triad as in FAS thioesterase (TE I). Mutating the conserved Asp2m or modifying it with Woodward's reagent K caused partial loss (40%) of TE II activity and reduced reactivity of Ser"l" and His237 toward their specific inhibitors, phenylmethylsulfonyl fluoride and diethylpyrocarbonate, respectively.These results suggested that Asp2m enhances, but is not essential for, the reactivity of Ser'l' and His237. Mutation analyses revealed that, at the C terminus, Leu262 is critical for TE II to interact with FAS. Hydrophobic interactions seem to play a role, since the interaction of TE H with FAS is enhanc by polyethylene glycol but reduced by salt. The Ser"°" and HiS237 mutants and a synthetic C-terminal decapeptide did not compete in the interaction. These results suggest that a TE Il-acyl FAS complex forms first, which then is stabilized by the interaction of the hydrophobic C terminus of TE H with FAS, leading ultimately to hydrolysis and release of fatty acid.Fatty acid synthase [FAS; acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl-and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85] catalyzes the synthesis of long-chain fatty acids from acetyl CoA, malonyl CoA, and NADPH. The native enzyme is a homodimer of a multifunctional protein (Mr, 274,510) (1, 2). Each protein has a site for the prosthetic group, 4'-phosphopantetheine (acyl carrier protein), to which the growing acyl groups are bound, and contains the six catalytic activities required for chain elongation and reduction and a thioesterase activity (TE I) for chain termination and release of free acid. The product of synthesis is primarily palmitate with stearate and myristate as minor components, each reflecting the substrate specificity of TE I for acyl CoA derivatives (2, 3). In some specialized tissues, such as the lactating mammary gland of nonruminant animals (4) and the uropygial gland of waterfowl (5), shorter chain acids (C8, C10, and C12) are also produced due to the presence of yet another thioesterase, medium-chain S-acyl FAS thioesterase (TE II), which is not part of FAS. TE II has broad substrate specificity and interacts with FAS to hydrolyze the fatty acyl thioesters before they mature to longerchain acids. The cDNA sequences of the TE Ils of the rat mammary gland (6) and of the uropygial gland of mallard ducks (7) EXPERIMENTAL PROCEDURESMaterials and Methods. FAS from rat livers (9) and native TE II from lactating rat mammary glands (10) were prepared as described.TE II was assayed by one or more of the foll...

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Molecular cloning and sequence analysis of complementary DNA encoding rat mammary gland medium-chain S-acyl fatty acid synthetase thio ester hydrolase

Cited by 32 publications

References 34 publications

Nucleotide sequence analysis reveals linked N-acetyl hydrolase, thioesterase, transport, and regulatory genes encoded by the bialaphos biosynthetic gene cluster of Streptomyces hygroscopicus

Nucleotide sequence analysis reveals linked N-acetyl hydrolase, thioesterase, transport, and regulatory genes encoded by the bialaphos biosynthetic gene cluster of Streptomyces hygroscopicus

Expression in Escherichia coli, purification and characterization of two mammalian thioesterases involved in fatty acid synthesis

Roles of Ser101, Asp236, and His237 in catalysis of thioesterase II and of the C-terminal region of the enzyme in its interaction with fatty acid synthase.

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