NDLI: Engineered Fatty Acid Biosynthesis in Streptomyces by Altered Catalytic Function of β-Ketoacyl-Acyl Carrier Protein Synthase III

Content Provider	PubMed Central
Author	Smirnova, Natalya Reynolds, Kevin A.
Copyright Year	2001
Abstract	The Streptomyces glaucescens β-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) initiates straight- and branched-chain fatty acid biosynthesis by catalyzing the decarboxylative condensation of malonyl-ACP with different acyl-coenzyme A (CoA) primers. This KASIII has one cysteine residue, which is critical for forming an acyl-enzyme intermediate in the first step of the process. Three mutants (Cys122Ala, Cys122Ser, Cys122Gln) were created by site-directed mutagenesis. Plasmid-based expression of these mutants in S. glaucescens resulted in strains which generated 75 (Cys122Ala) to 500% (Cys122Gln) more straight-chain fatty acids (SCFA) than the corresponding wild-type strain. In contrast, plasmid-based expression of wild-type KASIII had no effect on fatty acid profiles. These observations are attributed to an uncoupling of the condensation and decarboxylation activities in these mutants (malonyl-ACP is thus converted to acetyl-ACP, a SCFA precursor). Incorporation experiments with perdeuterated acetic acid demonstrated that 9% of the palmitate pool of the wild-type strain was generated from an intact D3 acetyl-CoA starter unit, compared to 3% in a strain expressing the Cys122Gln KASIII. These observations support the intermediacy of malonyl-ACP in generating the SCFA precursor in a strain expressing this mutant. To study malonyl-ACP decarboxylase activity in vitro, the KASIII mutants were expressed and purified as His-tagged proteins in Escherichia coli and assayed. In the absence of the acyl-CoA substrate the Cys122Gln mutant and wild-type KASIII were shown to have comparable decarboxylase activities in vitro. The Cys122Ala mutant exhibited higher activity. This activity was inhibited for all enzymes by the presence of high concentrations of isobutyryl-CoA (>100 μM), a branched-chain fatty acid biosynthetic precursor. Under these conditions the mutant enzymes had no activity, while the wild-type enzyme functioned as a ketoacyl synthase. These observations indicate the likely upper and lower limits of isobutyryl-CoA and related acyl-CoA concentrations within S. glaucescens.
Related Links	http://dx.doi.org/10.1128/jb.183.7.2335-2342.2001
Ending Page	2342
Page Count	8
Starting Page	2335
File Format	PDF
ISSN	00219193
e-ISSN	10985530
Journal	Journal of Bacteriology
Issue Number	7
Volume Number	183
Language	English
Publisher	American Society for Microbiology
Publisher Date	2001-04-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Molecular Biology Microbiology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Microbiology

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

β-Ketoacyl Acyl Carrier Protein Synthase III (FabH) Is Essential for Fatty Acid Biosynthesis in Streptomyces coelicolor A3(2)

Characterization of β-Ketoacyl-Acyl Carrier Protein Synthase III from Streptomyces glaucescens and Its Role in Initiation of Fatty Acid Biosynthesis

β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Is a Determining Factor in Branched-Chain Fatty Acid Biosynthesis

Crystal Structure of β-Ketoacyl-Acyl Carrier Protein Synthase III A KEY CONDENSING ENZYME IN BACTERIAL FATTY ACID BIOSYNTHESIS

Alteration of the Fatty Acid Profile of Streptomyces coelicolor by Replacement of the Initiation Enzyme 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH)

The role of β-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower

Improvement of fatty acid biosynthesis by engineered recombinant Escherichia coli

Fatty Acid Biosynthesis in Pseudomonas aeruginosa Is Initiated by the FabY Class of β-Ketoacyl Acyl Carrier Protein Synthases

Targeting the Fatty Acid Biosynthesis Enzyme, β-Ketoacyl–Acyl Carrier Protein Synthase III (PfKASIII), in the Identification of Novel Antimalarial Agents

Engineered Fatty Acid Biosynthesis in Streptomyces by Altered Catalytic Function of β-Ketoacyl-Acyl Carrier Protein Synthase III

Similar Documents

β-Ketoacyl Acyl Carrier Protein Synthase III (FabH) Is Essential for Fatty Acid Biosynthesis in Streptomyces coelicolor A3(2)

Characterization of β-Ketoacyl-Acyl Carrier Protein Synthase III from Streptomyces glaucescens and Its Role in Initiation of Fatty Acid Biosynthesis

β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Is a Determining Factor in Branched-Chain Fatty Acid Biosynthesis

Crystal Structure of β-Ketoacyl-Acyl Carrier Protein Synthase III A KEY CONDENSING ENZYME IN BACTERIAL FATTY ACID BIOSYNTHESIS

Alteration of the Fatty Acid Profile of Streptomyces coelicolor by Replacement of the Initiation Enzyme 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH)

The role of β-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower

Improvement of fatty acid biosynthesis by engineered recombinant Escherichia coli

Fatty Acid Biosynthesis in Pseudomonas aeruginosa Is Initiated by the FabY Class of β-Ketoacyl Acyl Carrier Protein Synthases

Targeting the Fatty Acid Biosynthesis Enzyme, β-Ketoacyl–Acyl Carrier Protein Synthase III (PfKASIII), in the Identification of Novel Antimalarial Agents

Engineered Fatty Acid Biosynthesis in Streptomyces by Altered Catalytic Function of β-Ketoacyl-Acyl Carrier Protein Synthase III