Partial purification and characterization of two enzymes involved in isovaleric acid synthesis in Clostridium bifermentans.
Conversion of leucine to isovaleric acid by Clostridium bifermentans is achieved by the action of at least two enzymes. One is a transaminase producing alpha-ketoisocaproic acid, which was purified 30-fold from osmotic lysates of late-exponential phase cells by repeated chromatography on DEAE-Sepharose C16B and Sephacryl S300: this represented a 147-fold purification of activity found in sonically disrupted cells. This enzyme had an apparent molecular weight of approximately 190000 and was composed of six identically sized sub-units (molecular weight 31000 +/- 1000). Transamination required pyridoxal phosphate and pyruvate and was optimal at pH 8.6; the apparent Km for leucine was 7.0 mM. Activity was totally inhibited by 1 mM-p-chloromercuribenzoate and partially inhibited by other thiol reagents. The second enzyme decarboxylated alpha-ketoisocaproic acid to form isovaleric acid and was also partially purified by chromatography on DEAE-Sepharose C16B and Sephacryl S300. It has an apparent molecular weight of 240000 and required FAD and coenzyme A for activity; the Km for alpha-ketoisocaproic acid was 4.2 mM and activity was optimal around pH 8.0. This enzyme was a flavoprotein with absorption maxima at 280, 320 and 400 nm, and a fluorescent maximum at 500 nm. The prosthetic group, FAD, dissociated from the protein during purification resulting in an inactive apoenzyme which was only partially re-activated by FAD. Activity was completely inhibited by several thiol reagents tested at 1 mM.
D002262:Carboxy-Lyases / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003013:Clostridium / Q000201:enzymology*; D003641:Deamination; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D045782:Hemiterpenes; D006863:Hydrogen-Ion Concentration; D007651:Keto Acids / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D008970:Molecular Weight; D010421:Pentanoic Acids / Q000096:biosynthesis*; D000637:Transaminases / Q000302:isolation & purification*; D014631:Valerates / Q000096:biosynthesis*
D045782:Hemiterpenes; D007651:Keto Acids; D010421:Pentanoic Acids; D014631:Valerates; D005182:Flavin-Adenine Dinucleotide; C008216:isovaleric acid; C013082:alpha-ketoisocaproic acid; D000637:Transaminases; D002262:Carboxy-Lyases; D007930:Leucine