Biochemical and genetic investigation of initial reactions in aerobic degradation of the bile acid cholate in Pseudomonas sp. strain Chol1.
Bile acids are surface-active steroid compounds with toxic effects for bacteria. Recently, the isolation and characterization of a bacterium, Pseudomonas sp. strain Chol1, growing with bile acids as the carbon and energy source was reported. In this study, initial reactions of the aerobic degradation pathway for the bile acid cholate were investigated on the biochemical and genetic level in strain Chol1. These reactions comprised A-ring oxidation, activation with coenzyme A (CoA), and beta-oxidation of the acyl side chain with the C(19)-steroid dihydroxyandrostadienedione as the end product. A-ring oxidizing enzyme activities leading to Delta(1,4)-3-ketocholyl-CoA were detected in cell extracts and confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cholate activation with CoA was demonstrated in cell extracts and confirmed with a chemically synthesized standard by LC-MS/MS. A transposon mutant with a block in oxidation of the acyl side chain accumulated a steroid compound in culture supernatants which was identified as 7alpha,12alpha-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) by nuclear magnetic resonance spectroscopy. The interrupted gene was identified as encoding a putative acyl-CoA-dehydrogenase (ACAD). DHOPDC activation with CoA in cell extracts of strain Chol1 was detected by LC-MS/MS. The growth defect of the transposon mutant could be complemented by the wild-type ACAD gene located on the plasmid pBBR1MCS-5. Based on these results, the initiating reactions of the cholate degradation pathway leading from cholate to dihydroxyandrostadienedione could be reconstructed. In addition, the first bacterial gene encoding an enzyme for a specific reaction step in side chain degradation of steroid compounds was identified, and it showed a high degree of similarity to genes in other steroid-degrading bacteria.
Birkenmaier
Antoinette
A
Universität Konstanz, Fachbereich Biologie, Mikrobielle Oekologie, Fach M654, D-78457, Konstanz, Germany.
Holert
Johannes
J
Erdbrink
Henrike
H
Moeller
Heiko M
HM
Friemel
Anke
A
Schoenenberger
René
R
Suter
Marc J-F
MJ
Klebensberger
Janosch
J
Philipp
Bodo
B
D042964:Acyl-CoA Dehydrogenase / Q000235:genetics; D000332:Aerobiosis; D000595:Amino Acid Sequence; D020355:Cholates / Q000378:metabolism*; D003065:Coenzyme A / Q000378:metabolism; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D017353:Gene Deletion; D005816:Genetic Complementation Test; D009682:Magnetic Resonance Spectroscopy; D053858:Metabolic Networks and Pathways / Q000235:genetics* / Q000502:physiology*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry
D020355:Cholates; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D042964:Acyl-CoA Dehydrogenase; D003065:Coenzyme A
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