Active multienzyme assemblies for long-chain olefinic hydrocarbon biosynthesis

James K Christenson, Matthew R Jensen, Brandon R. Goblirsch, Fatuma Mohamed, Wei Zhang, Carrie M Wilmot, Lawrence P Wackett

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Bacteria from different phyla produce long-chain olefinic hydrocarbons derived from an OleA-catalyzed Claisen condensation of two fatty acyl coenzyme A (acyl-CoA) substrates, followed by reduction and oxygen elimination reactions catalyzed by the proteins OleB, OleC, and OleD. In this report, OleA, OleB, OleC, and OleD were individually purified as soluble proteins, and all were found to be essential for reconstituting hydrocarbon biosynthesis. Recombinant coexpression of tagged OleABCD proteins from Xanthomonas campestris in Escherichia coli and purification over His6 and FLAG columns resulted in OleA separating, while OleBCD purified together, irrespective of which of the four Ole proteins were tagged. Hydrocarbon biosynthetic activity of copurified OleBCD assemblies could be reconstituted by adding separately purified OleA. Immunoblots of nondenaturing gels using anti-OleC reacted with X. campestris crude protein lysate indicated the presence of a large protein assembly containing OleC in the native host. Negative-stain electron microscopy of recombinant OleBCD revealed distinct large structures with diameters primarily between 24 and 40 nm. Assembling OleB, OleC, and OleD into a complex may be important to maintain stereochemical integrity of intermediates, facilitate the movement of hydrophobic metabolites between enzyme active sites, and protect the cell against the highly reactive β-lactone intermediate produced by the OleC-catalyzed reaction.

Original languageEnglish (US)
Article numbere00890-16
JournalJournal of bacteriology
Volume199
Issue number9
DOIs
StatePublished - May 1 2017

Fingerprint

Hydrocarbons
Olea
Xanthomonas campestris
Proteins
Acyl Coenzyme A
Lactones
Catalytic Domain
Electron Microscopy
Coloring Agents
Gels
Oxygen
Escherichia coli
Bacteria
Enzymes

Keywords

  • Bacteria
  • Hydrocarbon
  • Multienzyme complex
  • Olefin

Cite this

Active multienzyme assemblies for long-chain olefinic hydrocarbon biosynthesis. / Christenson, James K; Jensen, Matthew R; Goblirsch, Brandon R.; Mohamed, Fatuma; Zhang, Wei; Wilmot, Carrie M; Wackett, Lawrence P.

In: Journal of bacteriology, Vol. 199, No. 9, e00890-16, 01.05.2017.

Research output: Contribution to journalArticle

Christenson, James K ; Jensen, Matthew R ; Goblirsch, Brandon R. ; Mohamed, Fatuma ; Zhang, Wei ; Wilmot, Carrie M ; Wackett, Lawrence P. / Active multienzyme assemblies for long-chain olefinic hydrocarbon biosynthesis. In: Journal of bacteriology. 2017 ; Vol. 199, No. 9.
@article{9fb438067d8c45cd9899057ff1e0f338,
title = "Active multienzyme assemblies for long-chain olefinic hydrocarbon biosynthesis",
abstract = "Bacteria from different phyla produce long-chain olefinic hydrocarbons derived from an OleA-catalyzed Claisen condensation of two fatty acyl coenzyme A (acyl-CoA) substrates, followed by reduction and oxygen elimination reactions catalyzed by the proteins OleB, OleC, and OleD. In this report, OleA, OleB, OleC, and OleD were individually purified as soluble proteins, and all were found to be essential for reconstituting hydrocarbon biosynthesis. Recombinant coexpression of tagged OleABCD proteins from Xanthomonas campestris in Escherichia coli and purification over His6 and FLAG columns resulted in OleA separating, while OleBCD purified together, irrespective of which of the four Ole proteins were tagged. Hydrocarbon biosynthetic activity of copurified OleBCD assemblies could be reconstituted by adding separately purified OleA. Immunoblots of nondenaturing gels using anti-OleC reacted with X. campestris crude protein lysate indicated the presence of a large protein assembly containing OleC in the native host. Negative-stain electron microscopy of recombinant OleBCD revealed distinct large structures with diameters primarily between 24 and 40 nm. Assembling OleB, OleC, and OleD into a complex may be important to maintain stereochemical integrity of intermediates, facilitate the movement of hydrophobic metabolites between enzyme active sites, and protect the cell against the highly reactive β-lactone intermediate produced by the OleC-catalyzed reaction.",
keywords = "Bacteria, Hydrocarbon, Multienzyme complex, Olefin",
author = "Christenson, {James K} and Jensen, {Matthew R} and Goblirsch, {Brandon R.} and Fatuma Mohamed and Wei Zhang and Wilmot, {Carrie M} and Wackett, {Lawrence P}",
year = "2017",
month = "5",
day = "1",
doi = "10.1128/JB.00890-16",
language = "English (US)",
volume = "199",
journal = "Journal of Bacteriology",
issn = "0021-9193",
publisher = "American Society for Microbiology",
number = "9",

}

TY - JOUR

T1 - Active multienzyme assemblies for long-chain olefinic hydrocarbon biosynthesis

AU - Christenson, James K

AU - Jensen, Matthew R

AU - Goblirsch, Brandon R.

AU - Mohamed, Fatuma

AU - Zhang, Wei

AU - Wilmot, Carrie M

AU - Wackett, Lawrence P

PY - 2017/5/1

Y1 - 2017/5/1

N2 - Bacteria from different phyla produce long-chain olefinic hydrocarbons derived from an OleA-catalyzed Claisen condensation of two fatty acyl coenzyme A (acyl-CoA) substrates, followed by reduction and oxygen elimination reactions catalyzed by the proteins OleB, OleC, and OleD. In this report, OleA, OleB, OleC, and OleD were individually purified as soluble proteins, and all were found to be essential for reconstituting hydrocarbon biosynthesis. Recombinant coexpression of tagged OleABCD proteins from Xanthomonas campestris in Escherichia coli and purification over His6 and FLAG columns resulted in OleA separating, while OleBCD purified together, irrespective of which of the four Ole proteins were tagged. Hydrocarbon biosynthetic activity of copurified OleBCD assemblies could be reconstituted by adding separately purified OleA. Immunoblots of nondenaturing gels using anti-OleC reacted with X. campestris crude protein lysate indicated the presence of a large protein assembly containing OleC in the native host. Negative-stain electron microscopy of recombinant OleBCD revealed distinct large structures with diameters primarily between 24 and 40 nm. Assembling OleB, OleC, and OleD into a complex may be important to maintain stereochemical integrity of intermediates, facilitate the movement of hydrophobic metabolites between enzyme active sites, and protect the cell against the highly reactive β-lactone intermediate produced by the OleC-catalyzed reaction.

AB - Bacteria from different phyla produce long-chain olefinic hydrocarbons derived from an OleA-catalyzed Claisen condensation of two fatty acyl coenzyme A (acyl-CoA) substrates, followed by reduction and oxygen elimination reactions catalyzed by the proteins OleB, OleC, and OleD. In this report, OleA, OleB, OleC, and OleD were individually purified as soluble proteins, and all were found to be essential for reconstituting hydrocarbon biosynthesis. Recombinant coexpression of tagged OleABCD proteins from Xanthomonas campestris in Escherichia coli and purification over His6 and FLAG columns resulted in OleA separating, while OleBCD purified together, irrespective of which of the four Ole proteins were tagged. Hydrocarbon biosynthetic activity of copurified OleBCD assemblies could be reconstituted by adding separately purified OleA. Immunoblots of nondenaturing gels using anti-OleC reacted with X. campestris crude protein lysate indicated the presence of a large protein assembly containing OleC in the native host. Negative-stain electron microscopy of recombinant OleBCD revealed distinct large structures with diameters primarily between 24 and 40 nm. Assembling OleB, OleC, and OleD into a complex may be important to maintain stereochemical integrity of intermediates, facilitate the movement of hydrophobic metabolites between enzyme active sites, and protect the cell against the highly reactive β-lactone intermediate produced by the OleC-catalyzed reaction.

KW - Bacteria

KW - Hydrocarbon

KW - Multienzyme complex

KW - Olefin

UR - http://www.scopus.com/inward/record.url?scp=85017451115&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85017451115&partnerID=8YFLogxK

U2 - 10.1128/JB.00890-16

DO - 10.1128/JB.00890-16

M3 - Article

VL - 199

JO - Journal of Bacteriology

JF - Journal of Bacteriology

SN - 0021-9193

IS - 9

M1 - e00890-16

ER -