scholarly journals Terminal Olefin (1-Alkene) Biosynthesis by a Novel P450 Fatty Acid Decarboxylase fromJeotgalicoccusSpecies

2011 ◽  
Vol 77 (5) ◽  
pp. 1718-1727 ◽  
Author(s):  
Mathew A. Rude ◽  
Tarah S. Baron ◽  
Shane Brubaker ◽  
Murtaza Alibhai ◽  
Stephen B. Del Cardayre ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Catalytic Mechanism ◽  
Fatty Acid Biosynthesis ◽  
Draft Genome ◽  
Industrial Applications ◽  
Acid Decarboxylase ◽  
Cytochrome P450 Enzyme ◽  
Structural Determinants ◽  
Terminal Olefin ◽  
Terminal Olefins

ABSTRACTTerminal olefins (1-alkenes) are natural products that have important industrial applications as both fuels and chemicals. However, their biosynthesis has been largely unexplored. We describe a group of bacteria,Jeotgalicoccusspp., which synthesize terminal olefins, in particular 18-methyl-1-nonadecene and 17-methyl-1-nonadecene. These olefins are derived from intermediates of fatty acid biosynthesis, and the key enzyme inJeotgalicoccussp. ATCC 8456 is a terminal olefin-forming fatty acid decarboxylase. This enzyme,Jeotgalicoccussp. OleT (OleTJE), was identified by purification from cell lysates, and its encoding gene was identified from a draft genome sequence ofJeotgalicoccussp. ATCC 8456 using reverse genetics. Heterologous expression of the identified gene conferred olefin biosynthesis toEscherichia coli.OleTJEis a P450 from the cyp152 family, which includes bacterial fatty acid hydroxylases. Some cyp152 P450 enzymes have the ability to decarboxylate and to hydroxylate fatty acids (in α- and/or β-position), suggesting a common reaction intermediate in their catalytic mechanism and specific structural determinants that favor one reaction over the other. The discovery of these terminal olefin-forming P450 enzymes represents a third biosynthetic pathway (in addition to alkane and long-chain olefin biosynthesis) to convert fatty acid intermediates into hydrocarbons. Olefin-forming fatty acid decarboxylation is a novel reaction that can now be added to the catalytic repertoire of the versatile cytochrome P450 enzyme family.

scholarly journals Identification of active site residues implies a two-step catalytic mechanism for acyl-ACP thioesterase

2018 ◽  
Vol 475 (23) ◽  
pp. 3861-3873 ◽  
Author(s):  
Fuyuan Jing ◽  
Marna D. Yandeau-Nelson ◽  
Basil J. Nikolau
Keyword(s):  
Fatty Acid ◽  
Sequence Alignment ◽  
Fatty Acid Synthase ◽  
Catalytic Mechanism ◽  
Fatty Acid Biosynthesis ◽  
Tertiary Structure ◽  
Acyl Carrier Protein ◽  
Cocos Nucifera ◽  
Catalytic Residue ◽  
Thioester Bond

In plants and bacteria that use a Type II fatty acid synthase, isozymes of acyl-acyl carrier protein (ACP) thioesterase (TE) hydrolyze the thioester bond of acyl-ACPs, terminating the process of fatty acid biosynthesis. These TEs are therefore critical in determining the fatty acid profiles produced by these organisms. Past characterizations of a limited number of plant-sourced acyl-ACP TEs have suggested a thiol-based, papain-like catalytic mechanism, involving a triad of Cys, His, and Asn residues. In the present study, the sequence alignment of 1019 plant and bacterial acyl-ACP TEs revealed that the previously proposed Cys catalytic residue is not universally conserved and therefore may not be a catalytic residue. Systematic mutagenesis of this residue to either Ser or Ala in three plant acyl-ACP TEs, CvFatB1 and CvFatB2 from Cuphea viscosissima and CnFatB2 from Cocos nucifera, resulted in enzymatically active variants, demonstrating that this Cys residue (Cys348 in CvFatB2) is not catalytic. In contrast, the multiple sequence alignment, together with the structure modeling of CvFatB2, suggests that the highly conserved Asp309 and Glu347, in addition to previously proposed Asn311 and His313, may be involved in catalysis. The substantial loss of catalytic competence associated with site-directed mutants at these positions confirmed the involvement of these residues in catalysis. By comparing the structures of acyl-ACP TE and the Pseudomonas 4-hydroxybenzoyl-CoA TE, both of which fold in the same hotdog tertiary structure and catalyze the hydrolysis reaction of thioester bond, we have proposed a two-step catalytic mechanism for acyl-ACP TE that involves an enzyme-bound anhydride intermediate.

Binding of NADP+ triggers an open-to-closed transition in a mycobacterial FabG β-ketoacyl-ACP reductase

2017 ◽  
Vol 474 (6) ◽  
pp. 907-921 ◽  
Author(s):  
Mickaël Blaise ◽  
Niël Van Wyk ◽  
Françoise Banères-Roquet ◽  
Yann Guérardel ◽  
Laurent Kremer
Keyword(s):  
Fatty Acid ◽  
Rational Design ◽  
Fatty Acid Synthase ◽  
Catalytic Mechanism ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Reductase Activity ◽  
Protein A ◽  
Structural Features ◽  
Mycobacterial Growth

The ketoacyl-acyl carrier protein (ACP) reductase FabG catalyzes the NADPH/NADH dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products, the first reductive step in the fatty acid biosynthesis elongation cycle. FabG proteins are ubiquitous in bacteria and are part of the type II fatty acid synthase system. Mining the Mycobacterium smegmatis genome uncovered several putative FabG-like proteins. Among them, we identified M. smegmatis MSMEG_6753 whose gene was found adjacent to MSMEG_6754, encoding a recently characterized enoyl-CoA dehydratase, and to MSMEG_6755, encoding another potential reductase. Recombinantly expressed and purified MSMEG_6753 exhibits ketoacyl reductase activity in the presence of acetoacetyl-CoA and NADPH. This activity was subsequently confirmed by functional complementation studies in a fabG thermosensitive Escherichia coli mutant. Furthermore, comparison of the apo and the NADP+-bound MSMEG_6753 crystal structures showed that cofactor binding induces a closed conformation of the protein. A ΔMSMEG_6753 deletion mutant could be generated in M. smegmatis, indicating that this gene is dispensable for mycobacterial growth. Overall, these results showcase the diversity of FabG-like proteins in mycobacteria and new structural features regarding the catalytic mechanism of this important family of enzymes that may be of importance for the rational design of specific FabG inhibitors.

scholarly journals Draft genome of the liver fluke Fasciola gigantica

2018 ◽  
Author(s):  
Tripti Tripathi ◽  
Arpita Ghosh ◽  
Vivek N Todur ◽  
Parismita Kalita ◽  
R Vijayakumar ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Draft Genome ◽  
Fasciola Gigantica ◽  
Fatty Acid Production ◽  
Technological Advances ◽  
Food Borne ◽  
Liver Flukes ◽  
First Time

ABSTRACTFascioliasis is a neglected food-borne disease caused by liver flukes (genus Fasciola) and affects more than 200 million people worldwide. Despite technological advances, little is known about the molecular biology and biochemistry of the fluke. We present the draft genome of Fasciola gigantica for the first time. The assembled draft genome has a size of ~1.04 Gb with an N50 of 129 kb. A total of 20,858 genes were predicted. The de novo repeats identified in the draft genome were 46.85%. In pathway analysis, all the genes of glycolysis, Kreb’s cycle and fatty acid metabolism were found to be present, but the key genes for fatty acid production in fatty acid biosynthesis were missing. This indicates that the fatty acid required for the survival of the fluke may be acquired from the host bile. The genomic information will provide a comprehensive resource to facilitate the development of novel interventions for fascioliasis control.

scholarly journals Crystal structure of FabZ-ACP complex reveals a dynamic seesaw-like catalytic mechanism of dehydratase in fatty acid biosynthesis

Cell Research ◽  
2016 ◽  
Vol 26 (12) ◽  
pp. 1330-1344 ◽  
Author(s):  
Lin Zhang ◽  
Jianfeng Xiao ◽  
Jianrong Xu ◽  
Tianran Fu ◽  
Zhiwei Cao ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Fatty Acid ◽  
Catalytic Mechanism ◽  
Fatty Acid Biosynthesis ◽  
Acid Biosynthesis

Inhibition of early steps of de novo fatty-acid biosynthesis by different xenobiotica

1991 ◽  
Vol 81 (2) ◽  
pp. 251-255
Author(s):  
Manfred Focke ◽  
Andrea Feld ◽  
Hartmut K. Lichtenthaler
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Acid Biosynthesis

Development of 'S', 'N’ Heterocycles as Antimycobacterials Targeting Fatty Acid Biosynthesis

2019 ◽  
Vol 15 ◽  
Author(s):  
L. K. Dahiwade ◽  
S. R. Rochlani ◽  
P. B. Choudhari ◽  
R. P. Dhavale ◽  
H. N. Moreira
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Antimycobacterial Activity ◽  
Drug Candidate ◽  
Causative Organism ◽  
Present Communication ◽  
Acid Biosynthesis ◽  
Rational Development ◽  
After Cancer ◽  
Drug Resistant Strains

Background: Mycobacterium tuberculosis is a causative organism of tuberculosis, which is most deadly disease after cancer in a current decade. The development of multidrug and broadly drug- resistant strains making the tuberculosis problem more and more critical. In last 40 years, only one molecule is added to the treatment regimen. Generally, drug design and development programs are targeted proteins whose function is known to be essential to the bacterial cell. Objectives: Reported here are the development of 'S', 'N’ heterocycles as antimycobacterials targeting fatty acid biosynthesis. Material and Methods: In the present communication, rational development of anti-mycobacterial agent's targeting fatty acid biosynthesis has been done by integrating the pocket modelling and virtual analysis. Results: The identified potential 33 lead compounds were synthesized, characterized by physicochemical and spectroscopic methods like IR, NMR spectroscopy and further screened for antimycobacterial activity using isoniazid as standard. All the designed compounds have shown profound antimycobacterial activity. Conclusion: In this present communication, we found that 3c, 3f, 3l and 4k molecules had expressive desirable biological activity and specific interactions with fatty acids. Further optimization of these leads is necessary for the development of potential antimycobacterial drug candidate having less side effects.

scholarly journals Expression of novel acidic lipase from Micrococcus luteus in Pichia pastoris and its application in transesterification

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Selfela Restu Adina ◽  
Antonius Suwanto ◽  
Anja Meryandini ◽  
Esti Puspitasari
Keyword(s):  
Fatty Acid ◽  
Pichia Pastoris ◽  
Specific Activity ◽  
Signal Sequence ◽  
Micrococcus Luteus ◽  
Acid Methyl Ester ◽  
Industrial Applications ◽  
Expression Level ◽  
Lipase Gene ◽  
Acidic Lipase

Abstract Background Lipases are promising biocatalysts for industrial applications and attract attention to be explored. A novel acidic lipase has been isolated from the lipolytic bacteria Micrococcus luteus EMP48-D (LipEMP48-D) screened from tempeh. The lipase gene had previously been overexpressed in Escherichia coli BL21, but the expression level obtained was relatively low. Here, to improve the expression level, the lipase gene was cloned to Pichia pastoris. We eliminated the native signal sequence of M. luteus and replaced it with α-mating factor (α-MF) signal sequence. We also optimized and synthesized the lipase gene based on codon preference in P. pastoris. Results LipEMP48-D lipase was expressed as an extracellular protein. Codon optimization has been conducted for 20 codons, with the codon adaption index reaching 0.995. The highest extracellular lipase activity obtained reached 145.4 ± 4.8 U/mg under AOX1 promoter in P. pastoris KM71 strain, which was 9.7-fold higher than the previous activity in E. coli. LipEMP48-D showed the highest specific activity at pH 5.0 and stable within the pH range 3.0–5.0 at 40 °C. LipEMP48-D also has the capability of hydrolyzing various long-chain triglycerides, particularly olive oil (100%) followed by sunflower oil (88.5%). LipEMP48-D exhibited high tolerance for various polar organic solvents with low log P, such as isopropanol (115.7%) and butanol (114.6%). The metal ions (Na+, K+, Ca2+, Mg2+, Mn+) decreased enzyme activity up to 43.1%, while Fe2+ increased relative activity of enzymes up to 200%. The conversion of free fatty acid (FFA) into fatty acid methyl ester (FAME) was low around 2.95%. Conclusions This study was the first to report overexpression of Micrococcus lipase in yeast. The extracellular expression of this acidic lipase could be potential for biocatalyst in industrial fields, especially organic synthesis, food industry, and production of biodiesel.

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