12alpha- and 7alpha-hydroxysteroid dehydrogenase activities from Fusobacterium spp

Area of Science:

• Microbiology
• Biochemistry
• Enzymology

Background:

• Bile acid metabolism is critical in human health.
• Hydroxysteroid dehydrogenases (HSDH) play key roles in bile acid transformations.
• Screening fecal microbiota for novel HSDH activities is essential for understanding these pathways.

Purpose of the Study:

• To isolate and characterize microorganisms possessing hydroxysteroid dehydrogenase activities relevant to bile acid metabolism.
• To identify specific HSDH isozymes and their catalytic properties.

Main Methods:

• Screening of fecal anaerobic organisms.
• Isolation of a gram-negative, Bacteroides-like anaerobe.
• Enzyme assays for 12alpha- and 7alpha-hydroxysteroid dehydrogenase (HSDH) activities.
• Enzyme characterization using stability tests, Sephadex G 200 chromatography, and thin-layer chromatography.
• Preliminary kinetic studies of 12alpha-HSDH.

Main Results:

• Isolation of a novel Bacteroides-like anaerobe exhibiting both 12alpha-HSDH and 7alpha-HSDH activities.
• Quantification of enzyme production: approximately 4500 units 12alpha-HSDH and 360 units 7alpha-HSDH per 10(10) viable cells.
• Evidence suggesting the two HSDH activities are from separate enzymes based on stability and chromatographic behavior.
• Confirmation of oxidation at 12alpha-OH and 7alpha-OH positions, with no measurable 3alpha-OH activity.
• 12alpha-HSDH optimal activity at pH 9.5, with Michaelis constants for glycodeoxycholate and NADP determined.

Conclusions:

• A novel Bacteroides-like anaerobe possesses distinct 12alpha- and 7alpha-hydroxysteroid dehydrogenase activities.
• These enzymes are potentially valuable for studying bile acid metabolism.
• Further characterization of these HSDH enzymes could reveal new insights into microbial bile acid biotransformation.