scholarly journals Pathway to Synthesis and Processing of Mycolic Acids in Mycobacterium tuberculosis

2005 ◽  
Vol 18 (1) ◽  
pp. 81-101 ◽  
Author(s):  
Kuni Takayama ◽  
Cindy Wang ◽  
Gurdyal S. Besra
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Fatty Acid ◽  
Candidate Genes ◽  
Abc Transporter ◽  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Fatty Acid Synthetase ◽  
Mycolic Acids ◽  
Trehalose Dimycolate ◽  
Trehalose Monomycolate

SUMMARY Mycobacterium tuberculosis is known to synthesize α-, methoxy-, and keto-mycolic acids. We propose a detailed pathway to the biosynthesis of all mycolic acids in M. tuberculosis. Fatty acid synthetase I provides C20-S-coenzyme A to the fatty acid synthetase II system (FAS-IIA). Modules of FAS-IIA and FAS-IIB introduce cis unsaturation at two locations on a growing meroacid chain to yield three different forms of cis,cis-diunsaturated fatty acids (intermediates to α-, methoxy-, and keto-meroacids). These are methylated, and the mature meroacids and carboxylated C26-S-acyl carrier protein enter into the final Claisen-type condensation with polyketide synthase-13 (Pks13) to yield mycolyl-S-Pks13. We list candidate genes in the genome encoding the proposed dehydrase and isomerase in the FAS-IIA and FAS-IIB modules. We propose that the processing of mycolic acids begins by transfer of mycolic acids from mycolyl-S-Pks13 to d-mannopyranosyl-1-phosphoheptaprenol to yield 6-O-mycolyl-β-d-mannopyranosyl-1-phosphoheptaprenol and then to trehalose 6-phosphate to yield phosphorylated trehalose monomycolate (TMM-P). Phosphatase releases the phosphate group to yield TMM, which is immediately transported outside the cell by the ABC transporter. Antigen 85 then catalyzes the transfer of a mycolyl group from TMM to the cell wall arabinogalactan and to other TMMs to produce arabinogalactan-mycolate and trehalose dimycolate, respectively. We list candidate genes in the genome that encode the proposed mycolyltransferases I and II, phosphatase, and ABC transporter. The enzymes within this total pathway are targets for new drug discovery.

scholarly journals Revisiting the Assignment of Rv0241c to Fatty Acid Synthase Type II of Mycobacterium tuberculosis

2010 ◽  
Vol 192 (15) ◽  
pp. 4037-4044 ◽  
Author(s):  
Emmanuelle Sacco ◽  
Nawel Slama ◽  
Kristina Bäckbro ◽  
Tanya Parish ◽  
Françoise Laval ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Mycolic Acid ◽  
Type Ii ◽  
Heterologous Complementation ◽  
Mycolic Acids ◽  
Fas Ii

ABSTRACT The fatty acid synthase type II enzymatic complex of Mycobacterium tuberculosis (FAS-II Mt ) catalyzes an essential metabolic pathway involved in the biosynthesis of major envelope lipids, mycolic acids. The partner proteins of this singular FAS-II system represent relevant targets for antituberculous drug design. Two heterodimers of the hydratase 2 protein family, HadAB and HadBC, were shown to be involved in the (3R)-hydroxyacyl-ACP dehydration (HAD) step of FAS-II Mt cycles. Recently, an additional member of this family, Rv0241c, was proposed to have the same function, based on the heterologous complementation of a HAD mutant of the yeast mitochondrial FAS-II system. In the present work, Rv0241c was able to complement a HAD mutant in the Escherichia coli model but not a dehydratase-isomerase deficient mutant. However, an enzymatic study of the purified protein demonstrated that Rv0241c possesses a broad chain length specificity for the substrate, unlike FAS-II Mt enzymes. Most importantly, Rv0241c exhibited a strict dependence on the coenzyme A (CoA) as opposed to AcpM, the natural acyl carrier protein bearing the chains elongated by FAS-II Mt . The deletion of Rv0241c showed that this gene is not essential to M. tuberculosis survival in vitro. The resulting mutant did not display any change in the mycolic acid profile. This demonstrates that Rv0241c is a trans-2-enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydratase that does not belong to FAS-II Mt . The relevance of a heterologous complementation strategy to identifying proteins of such a system is questioned.

scholarly journals A Futile Metabolic Cycle of Fatty Acyl-CoA Hydrolysis and Resynthesis in Corynebacterium glutamicum and Its Disruption Leading to Fatty Acid Production

2020 ◽  
Author(s):  
Masato Ikeda ◽  
Keisuke Takahashi ◽  
Tatsunori Ohtake ◽  
Ryosuke Imoto ◽  
Haruka Kawakami ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Free Fatty Acids ◽  
Corynebacterium Glutamicum ◽  
Candidate Genes ◽  
Outer Layer ◽  
Acid Production ◽  
Fatty Acyl ◽  
Fatty Acid Production ◽  
Mycolic Acids

Fatty acyl-CoA thioesterase (Tes) and acyl-CoA synthetase (FadD) catalyze opposing reactions between acyl-CoAs and free fatty acids. Within the genome of Corynebacterium glutamicum, several candidate genes for each enzyme are present, although their functions remain unknown. Modified expressions of the candidate genes in the fatty acid producer WTΔfasR led to identification of one tes gene (tesA) and two fadD genes (fadD5 and fadD15), which functioned positively and negatively in fatty acid production, respectively. Genetic analysis showed that fadD5 and fadD15 are responsible for utilization of exogenous fatty acids and that tesA plays a role in supplying fatty acids for synthesis of the outer layer components mycolic acids. Enzyme assays and expression analysis revealed that tesA, fadD5, and fadD15 were co-expressed to create a cyclic route between acyl-CoAs and fatty acids. When fadD5 or fadD15 was disrupted in wild-type C. glutamicum, both disruptants excreted fatty acids during growth. Double disruptions of them resulted in a synergistic increase in production. Additional disruption of tesA revealed a canceling effect on production. These results indicate that the FadDs normally shunt the surplus of TesA-generated fatty acids back to acyl-CoAs for lipid biosynthesis and that interception of this shunt provokes cells to overproduce fatty acids. When this strategy was applied to a fatty acid high-producer, the resulting fadDs-disrupted and tesA-amplified strain exhibited a 72% yield increase relative to its parent and produced fatty acids, which consisted mainly of oleic acid, palmitic acid, and stearic acid, on the gram scale per liter from 1% glucose. IMPORTANCE The industrial amino acid producer Corynebacterium glutamicum has currently evolved into a potential workhorse for fatty acid production. In this organism, we obtained evidence showing the presence of a unique mechanism of lipid homeostasis, namely, a formation of a futile cycle of acyl-CoA hydrolysis and resynthesis mediated by acyl-CoA thioesterase (Tes) and acyl-CoA synthetase (FadD), respectively. The biological role of the coupling of Tes and FadD would be to supply free fatty acids for synthesis of the outer layer components mycolic acids and to recycle their surplusage to acyl-CoAs for membrane lipid synthesis. We further demonstrated that engineering of the cycle in a fatty acid high-producer led to dramatically improved production, which provides a useful engineering strategy for fatty acid production in this industrially important microorganism.

scholarly journals Active site labeling of fatty acid and polyketide acyl-carrier protein transacylases

2019 ◽  
Vol 17 (19) ◽  
pp. 4720-4724 ◽  
Author(s):  
Tony D. Davis ◽  
Jennifer M. Michaud ◽  
Michael D. Burkart
Keyword(s):  
Fatty Acid ◽  
Fluorescent Probe ◽  
Active Site ◽  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Site Directed Mutagenesis ◽  
Probe Design ◽  
Directed Mutagenesis ◽  
Carrier Protein

Fluorescent probe design and site-directed mutagenesis unveil new activity-based chemical reporters for fatty acid and polyketide synthase acyl-carrier protein transacylases.

scholarly journals Mn(III) Pyrophosphate as an Efficient Tool for Studying the Mode of Action of Isoniazid on the InhA Protein of Mycobacterium tuberculosis

2002 ◽  
Vol 46 (7) ◽  
pp. 2137-2144 ◽  
Author(s):  
Michel Nguyen ◽  
Annaïk Quémard ◽  
Sylvain Broussy ◽  
Jean Bernadou ◽  
Bernard Meunier
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Biosynthetic Pathway ◽  
Acyl Carrier Protein ◽  
Isonicotinic Acid ◽  
Structural Investigations ◽  
Mycolic Acids ◽  
Nicotinamide Mononucleotide ◽  
Catalase Peroxidase ◽  
Nicotinamide Coenzymes

ABSTRACT The antituberculosis drug isoniazid (INH) is quickly oxidized by stoichiometric amounts of manganese(III) pyrophosphate. In the presence of nicotinamide coenzymes (NAD+, NADH, nicotinamide mononucleotide [NMN+]) and nicotinic acid adenine dinucleotide (DNAD+), INH oxidation produced the formation of INH-coenzyme adducts in addition to known biologically inactive products (isonicotinic acid, isonicotinamide, and isonicotinaldehyde). A pool of INH-NAD(H) adducts preformed in solution allowed the rapid and strong inhibition of in vitro activity of the enoyl-acyl carrier protein reductase InhA, an INH target in the biosynthetic pathway of mycolic acids: the inhibition was 90 or 60% when the adducts were formed in the presence of NAD+ or NADH, respectively. Under similar conditions, no inhibitory activity of INH-NMN(H) and INH-DNAD(H) adducts was detected. When an isolated pool of 100 nM INH-NAD(H) adducts was first incubated with InhA, the enzyme activity was inhibited by 80%; when present in excess, both NADH and decenoyl-coenzyme A are able to prevent this phenomenon. InhA inhibition by several types of INH-coenzyme adducts coexisting in solution is discussed in relation with the structure of the coenzyme, the stereochemistry of the adducts, and their existence as both open and cyclic forms. Thus, manganese(III) pyrophosphate appears to be an efficient and convenient alternative oxidant to mimic the activity of the Mycobacterium tuberculosis KatG catalase-peroxidase and will be useful for further mechanistic studies of INH activation and for structural investigations of reactive INH species in order to promote the design of new inhibitors of InhA as potential antituberculous drugs.

scholarly journals Purification and Biochemical Characterization of theMycobacterium tuberculosisβ-Ketoacyl-acyl Carrier Protein Synthases KasA and KasB

2001 ◽  
Vol 276 (50) ◽  
pp. 47029-47037 ◽  
Author(s):  
Merrill L. Schaeffer ◽  
Gautam Agnihotri ◽  
Craig Volker ◽  
Howard Kallender ◽  
Patrick J. Brennan ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Carrier Protein ◽  
Mycolic Acids ◽  
E Coli ◽  
Long Chain

Mycolic acids are vital components of theMycobacterium tuberculosiscell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performsde novofatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two β-ketoacyl-ACP synthase (KAS) enzymes of theM. tuberculosisFASII system. KasA and KasB were expressed inE. coliand purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use ofM. tuberculosisAcpM, suggesting that structural differences between AcpM andE. coliACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery.

Isolation and sequencing of a genomic DNA clone containing the 3′ terminus of the 6-methylsalicylic acid polyketide synthetase gene of Penicillium urticae

1991 ◽  
Vol 37 (1) ◽  
pp. 86-95 ◽  
Author(s):  
Ing-Kae Wang ◽  
C. Reeves ◽  
G. M. Gaucher
Keyword(s):  
Amino Acids ◽  
Fatty Acid ◽  
Amino Acid ◽  
Genomic Dna ◽  
Active Sites ◽  
Acyl Carrier Protein ◽  
Fatty Acid Synthetase ◽  
Protein Domain ◽  
Nucleotide Sequence Analysis ◽  
Functional Domains

A 7.7-kilobase (kb) Penicillium urticae genomic DNA fragment containing the 3′ terminus of the 6-methylsalicylic acid polyketide synthetase gene was cloned using a 41-mer mixed oligodeoxynucleotide probe which was based on a cyanogen bromide cleavage peptide of 35 amino acids obtained from pure synthetase. Nucleotide sequence analysis of a 2.2-kb region of the cloned fragment revealed a large open reading frame of 1866 bases which was devoid of introns and which corresponded to amino acids of the carboxyl terminus of the enzyme. This was followed by a putative transcription termination–polyadenylation signal. A putative acyl carrier protein domain at the 3′ terminus was preceded by a β-ketoreductase domain. These functionalities were identified by amino acid sequences known to be characteristic of the active sites of fatty acid synthetase functional domains. Their relative positions contrast with those in yeast and P. urticae fatty acid synthetase genes where the two functional domains are located at the 5′ terminus and in reverse order. Furthermore, amino acid sequence identities indicated a striking homology with vertebrate rather than either yeast or P. urticae fatty acid synthetases. Key words: multifunctional enzyme, mycotoxin, Pencillium, polyketide synthetase, secondary metabolism.

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