scholarly journals Development and retention of a primordial moonlighting pathway of protein modification in the absence of selection presents a puzzle

2018 ◽  
Vol 115 (4) ◽  
pp. 647-655 ◽  
Author(s):  
Xinyun Cao ◽  
Yaoqin Hong ◽  
Lei Zhu ◽  
Yuanyuan Hu ◽  
John E. Cronan
Keyword(s):  
Lipoic Acid ◽  
Protein Modification ◽  
Acid Synthesis ◽  
Covalent Attachment ◽  
Aquifex Aeolicus ◽  
Original Function ◽  
E Coli ◽  
Metabolism Enzymes ◽  
Glycine Cleavage ◽  
H Protein

Lipoic acid is synthesized by a remarkably atypical pathway in which the cofactor is assembled on its cognate proteins. An octanoyl moiety diverted from fatty acid synthesis is covalently attached to the acceptor protein, and sulfur insertion at carbons 6 and 8 of the octanoyl moiety form the lipoyl cofactor. Covalent attachment of this cofactor is required for function of several central metabolism enzymes, including the glycine cleavage H protein (GcvH). InBacillus subtilis, GcvH is the sole substrate for lipoate assembly. Hence lipoic acid-requiring 2-oxoacid dehydrogenase (OADH) proteins acquire the cofactor only by transfer from lipoylated GcvH. Lipoyl transfer has been argued to be the primordial pathway of OADH lipoylation. TheEscherichia colipathway where lipoate is directly assembled on both its GcvH and OADH proteins, is proposed to have arisen later. Because roughly 3 billion years separate the divergence of these bacteria, it is surprising thatE. coliGcvH functionally substitutes for theB. subtilisprotein in lipoyl transfer. Known and putative GcvHs from other bacteria and eukaryotes also substitute forB. subtilisGcvH in OADH modification. Because glycine cleavage is the primary GcvH role in ancestral bacteria that lack OADH enzymes, lipoyl transfer is a “moonlighting” function: that is, development of a new function while retaining the original function. This moonlighting has been conserved in the absence of selection by some, but not all, GcvH proteins. Moreover,Aquifex aeolicusencodes five putative GcvHs, two of which have the moonlighting function, whereas others function only in glycine cleavage.

scholarly journals A Novel Lipoate-Protein Ligase, Mhp-LplJ, Is Required for Lipoic Acid Metabolism in Mycoplasma hyopneumoniae

2021 ◽  
Vol 11 ◽  
Author(s):  
Jin Jin ◽  
Huan Chen ◽  
Ning Wang ◽  
Kemeng Zhu ◽  
Huanhuan Liu ◽  
...  
Keyword(s):  
Lipoic Acid ◽  
Acid Metabolism ◽  
Model Organism ◽  
Mycoplasma Hyopneumoniae ◽  
Vital Role ◽  
Metabolism Enzymes ◽  
Glycine Cleavage ◽  
One Carbon Metabolism ◽  
Enzyme Complexes

Lipoic acid is a conserved cofactor necessary for the activation of several critical enzyme complexes in the aerobic metabolism of 2-oxoacids and one-carbon metabolism. Lipoate metabolism enzymes are key for lipoic acid biosynthesis and salvage. In this study, we found that Mycoplasma hyopneumoniae (M. hyopneumoniae) Mhp-Lpl, which had been previously shown to have lipoate-protein ligase activity against glycine cleavage system H protein (GcvH) in vitro, did not lipoylate the lipoate-dependent subunit of dihydrolipoamide dehydrogenase (PdhD). Further studies indicated that a new putative lipoate-protein ligase in M. hyopneumoniae, MHP_RS00640 (Mhp-LplJ), catalyzes free lipoic acid attachment to PdhD in vitro. In a model organism, Mhp-LplJ exhibited lipoate and octanoate ligase activities against PdhD. When the enzyme activity of Mhp-LplJ was disrupted by lipoic acid analogs, 8-bromooctanoic acid (8-BrO) and 6,8-dichlorooctanoate (6,8-diClO), M. hyopneumoniae growth was arrested in vitro. Taken together, these results indicate that Mhp-LplJ plays a vital role in lipoic acid metabolism of M. hyopneumoniae, which is of great significance to further understand the metabolism of M. hyopneumoniae and develop new antimicrobials against it.

scholarly journals Protein moonlighting elucidates the essential human pathway catalyzing lipoic acid assembly on its cognate enzymes

2018 ◽  
Vol 115 (30) ◽  
pp. E7063-E7072 ◽  
Author(s):  
Xinyun Cao ◽  
Lei Zhu ◽  
Xuejiao Song ◽  
Zhe Hu ◽  
John E. Cronan
Keyword(s):  
Lipoic Acid ◽  
Metabolic Disorders ◽  
Enzyme Level ◽  
Early Death ◽  
Glycine Cleavage System ◽  
Amino Acid Degradation ◽  
Group Assembly ◽  
Glycine Cleavage ◽  
Assembly Map ◽  
H Protein

The lack of attachment of lipoic acid to its cognate enzyme proteins results in devastating human metabolic disorders. These mitochondrial disorders are evident soon after birth and generally result in early death. The mutations causing specific defects in lipoyl assembly map in three genes, LIAS, LIPT1, and LIPT2. Although physiological roles have been proposed for the encoded proteins, only the LIPT1 protein had been studied at the enzyme level. LIPT1 was reported to catalyze only the second partial reaction of the classical lipoate ligase mechanism. We report that the physiologically relevant LIPT1 enzyme activity is transfer of lipoyl moieties from the H protein of the glycine cleavage system to the E2 subunits of the 2-oxoacid dehydrogenases required for respiration (e.g., pyruvate dehydrogenase) and amino acid degradation. We also report that LIPT2 encodes an octanoyl transferase that initiates lipoyl group assembly. The human pathway is now biochemically defined.

scholarly journals Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway

2016 ◽  
Vol 80 (2) ◽  
pp. 429-450 ◽  
Author(s):  
John E. Cronan
Keyword(s):  
Lipoic Acid ◽  
Biosynthetic Pathway ◽  
Sequence Similarity ◽  
Pfam Family ◽  
Glycine Cleavage System ◽  
Content Type ◽  
Metabolism Enzymes ◽  
Glycine Cleavage ◽  
State Of Affairs ◽  
Eukaryotic Organisms

SUMMARYAlthough the structure of lipoic acid and its role in bacterial metabolism were clear over 50 years ago, it is only in the past decade that the pathways of biosynthesis of this universally conserved cofactor have become understood. Unlike most cofactors, lipoic acid must be covalently bound to its cognate enzyme proteins (the 2-oxoacid dehydrogenases and the glycine cleavage system) in order to function in central metabolism. Indeed, the cofactor is assembled on its cognate proteins rather than being assembled and subsequently attached as in the typical pathway, like that of biotin attachment. The first lipoate biosynthetic pathway determined was that ofEscherichia coli, which utilizes two enzymes to form the active lipoylated protein from a fatty acid biosynthetic intermediate. Recently, a more complex pathway requiring four proteins was discovered inBacillus subtilis, which is probably an evolutionary relic. This pathway requires the H protein of the glycine cleavage system of single-carbon metabolism to form active (lipoyl) 2-oxoacid dehydrogenases. The bacterial pathways inform the lipoate pathways of eukaryotic organisms. Plants use theE. colipathway, whereas mammals and fungi probably use theB. subtilispathway. The lipoate metabolism enzymes (except those of sulfur insertion) are members of PFAM family PF03099 (the cofactor transferase family). Although these enzymes share some sequence similarity, they catalyze three markedly distinct enzyme reactions, making the usual assignment of function based on alignments prone to frequent mistaken annotations. This state of affairs has possibly clouded the interpretation of one of the disorders of human lipoate metabolism.

Glycine cleavage enzyme complex: Molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts

2011 ◽  
Vol 89 (3) ◽  
pp. 299-307 ◽  
Author(s):  
Agnes Zay ◽  
Francis Y.M. Choy ◽  
Chelsea Patrick ◽  
Graham Sinclair
Keyword(s):  
Pichia Pastoris ◽  
Human Skin ◽  
Lipoic Acid ◽  
Culture Medium ◽  
Skin Fibroblasts ◽  
Enzyme Complex ◽  
Human Skin Fibroblasts ◽  
Cleavage Enzyme ◽  
Glycine Cleavage ◽  
H Protein

The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to interact with the L-protein, which is also part of the l-ketoglutarate dehydrogenase complex present in fibroblasts.

scholarly journals Genetic dissection of the mitochondrial lipoylation pathway in yeast

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Laura P. Pietikäinen ◽  
M. Tanvir Rahman ◽  
J. Kalervo Hiltunen ◽  
Carol L. Dieckmann ◽  
Alexander J. Kastaniotis
Keyword(s):  
Lipoic Acid ◽  
Model Organism ◽  
Acid Synthesis ◽  
Fatty Acyl ◽  
Model System ◽  
Genetic Dissection ◽  
Post Translational Modification ◽  
Mitochondrial Fatty Acid ◽  
Glycine Cleavage

Abstract Background Lipoylation of 2-ketoacid dehydrogenases is essential for mitochondrial function in eukaryotes. While the basic principles of the lipoylation processes have been worked out, we still lack a thorough understanding of the details of this important post-translational modification pathway. Here we used yeast as a model organism to characterize substrate usage by the highly conserved eukaryotic octanoyl/lipoyl transferases in vivo and queried how amenable the lipoylation system is to supplementation with exogenous substrate. Results We show that the requirement for mitochondrial fatty acid synthesis to provide substrates for lipoylation of the 2-ketoacid dehydrogenases can be bypassed by supplying the cells with free lipoic acid (LA) or octanoic acid (C8) and a mitochondrially targeted fatty acyl/lipoyl activating enzyme. We also provide evidence that the S. cerevisiae lipoyl transferase Lip3, in addition to transferring LA from the glycine cleavage system H protein to the pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGD) E2 subunits, can transfer this cofactor from the PDH complex to the KGD complex. In support of yeast as a model system for human metabolism, we demonstrate that the human octanoyl/lipoyl transferases can substitute for their counterparts in yeast to support respiratory growth and protein lipoylation. Like the wild-type yeast enzyme, the human lipoyl transferase LIPT1 responds to LA supplementation in the presence of the activating enzyme LplA. Conclusions In the yeast model system, the eukaryotic lipoylation pathway can use free LA and C8 as substrates when fatty/lipoic acid activating enzymes are targeted to mitochondria. Lip3 LA transferase has a wider substrate specificity than previously recognized. We show that these features of the lipoylation mechanism in yeast are conserved in mammalian mitochondria. Our findings have important implications for the development of effective therapies for the treatment of LA or mtFAS deficiency-related disorders.

scholarly journals Genetic dissection of the mitochondrial lipoylation pathway in yeast

2020 ◽  
Author(s):  
Laura P. Pietikäinen ◽  
M. Tanvir Rahman ◽  
J. Kalervo Hiltunen ◽  
Carol L. Dieckmann ◽  
Alexander J. Kastaniotis
Keyword(s):  
Lipoic Acid ◽  
Model Organism ◽  
Acid Synthesis ◽  
Fatty Acyl ◽  
Model System ◽  
Genetic Dissection ◽  
Post Translational Modification ◽  
Mitochondrial Fatty Acid ◽  
Glycine Cleavage

ABSTRACTBackgroundLipoylation of 2-ketoacid dehydrogenases is essential for mitochondrial function in eukaryotes. While the basic principles of the lipoylation processes have been worked out, we still lack a thorough understanding of the details of this important post-translational modification pathway. Here we used yeast as a model organism to characterize substrate usage by the highly conserved eukaryotic octanoyl/lipoyl transferases in vivo and queried how amenable the lipoylation system is to supplementation with exogenous substrate.ResultsWe show that the requirement for mitochondrial fatty acid synthesis to provide substrates for lipoylation of the 2-ketoacid dehydrogenases can be bypassed by supplying the cells with free lipoic acid (LA) or octanoic acid (C8) and a mitochondrially targeted fatty acyl/lipoyl activating enzyme. We also provide evidence that the S. cerevisiae lipoyl transferase Lip3, in addition to transferring LA from the glycine cleavage system H protein to the pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGD) E2 subunits, can transfer this cofactor from the PDH complex to the KGD complex. In support of yeast as a model system for human metabolism, we demonstrate that the human octanoyl/lipoyl transferases can substitute for their counterparts in yeast to support respiratory growth and protein lipoylation. Like the wild-type yeast enzyme, the human lipoyl transferase LIPT1 responds to LA supplementation in the presence of the activating enzyme LplA.ConclusionsIn the yeast model system, the eukaryotic lipoylation pathway can use free LA and C8 as substrates when fatty/lipoic acid activating enzymes are targeted to mitochondria. Lip3 LA transferase has a wider substrate specificity than previously recognized. We show that these features of the lipoylation mechanism in yeast are conserved in mammalian mitochondria. Our findings have important implications for the development of effective therapies for the treatment of LA or mtFAS deficiency-related disorders.

scholarly journals Biosynthesis of the Cyanobacterial Light-Harvesting Polypeptide Phycoerythrocyanin Holo-α Subunit in a Heterologous Host

2002 ◽  
Vol 184 (17) ◽  
pp. 4666-4671 ◽  
Author(s):  
Aaron J. Tooley ◽  
Alexander N. Glazer
Keyword(s):  
Ternary Complexes ◽  
Covalent Attachment ◽  
Heme Oxygenase 1 ◽  
Α Subunit ◽  
Ionization Time ◽  
E Coli ◽  
Metal Affinity ◽  
Flight Mass Spectrometry ◽  
Genes Encoding ◽  
Cyanobacterial Genes

ABSTRACT The entire pathway for the biosynthesis of the phycobiliviolin-bearing His-tagged holo-α subunit of the cyanobacterial photosynthetic accessory protein phycoerythrocyanin was reconstituted in Escherichia coli. Cyanobacterial genes encoding enzymes required for the conversion of heme to 3Z-phycocyanobilin, a precursor of phycobiliviolin (namely, heme oxygenase 1 and 3Z-phycocyanobilin:ferredoxin oxidoreductase), were expressed from a plasmid under the control of the hybrid trp-lac (trc) promoter. Genes for the apo-phycoerythrocyanin α subunit (pecA) and the heterodimeric lyase/isomerase (pecE and pecF), which catalyzes both the covalent attachment of phycocyanobilin and its concurrent isomerization to phycobiliviolin, were expressed from the trc promoter on a second plasmid. Upon induction, recombinant E. coli used endogenous heme to produce holo-PecA with absorbance and fluorescence properties similar to those of the same protein produced in cyanobacteria. About two-thirds of the apo-PecA was converted to holo-PecA. No significant bilin addition took place in a similarly engineered E. coli strain that lacks pecE and pecF. By using immobilized metal affinity chromatography, both apo-PecA and holo-PecA were isolated as ternary complexes with PecE and PecF. The identities of all three components in the ternary complexes were established unambiguously by protein and tryptic peptide analyses performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

scholarly journals Induction and control of the autolytic system of Escherichia coli

1982 ◽  
Vol 152 (1) ◽  
pp. 26-34
Author(s):  
M Leduc ◽  
R Kasra ◽  
J van Heijenoort
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Acid Synthesis ◽  
Induction Method ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Carbonyl Cyanide ◽  
And Control ◽  
First Time ◽  
Induced Autolysis

Various methods of inducing autolysis of Escherichia coli cells were investigated, some being described here for the first time. For the autolysis of growing cells only induction methods interfering with the biosynthesis of peptidoglycan were taken into consideration, whereas with harvested cells autolysis was induced by rapid osmotic or EDTA shock treatments. The highest rates of autolysis were observed after induction by moenomycin, EDTA, or cephaloridine. The different autolyses examined shared certain common properties. In particular, regardless of the induction method used, more or less extensive peptidoglycan degradation was observed, and 10(-2) M Mg2+ efficiently inhibited the autolytic process. However, for other properties a distinction was made between methods used for growing cells and those used for harvested cells. Autolysis of growing cells required RNA, protein, and fatty acid synthesis. No such requirements were observed with shock-induced autolysis performed with harvested cells. Thus, the effects of Mg2+, rifampicin, chloramphenicol, and cerulenin clearly suggest that distinct factors are involved in the control of the autolytic system of E. Coli. Uncoupling agents such as sodium azide, 2,4-dinitrophenol, and carbonyl-cyanide-m-chlorophenyl hydrazone used at their usual inhibiting concentration had no effect on the cephaloridine or shock-induced autolysis.

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