Enzymatic reactions in the degradation of 5-aminovalerate by Clostridium aminovalericum.

The anaerobic degradation of 5-aminovalerate to valerate, acetate, propionate, and ammonia by Clostridium aminovalericum was shown to involve the following intermediates: glutaric semialdehyde, 5-hydroxyvalerate, 5-hydroxyvaleryl-CoA, 4-pentenoyl-CoA, 2,4-pentadienoyl-CoA, trans-2-pentenoyl-CoA, L-3-hydroxyvaleryl-CoA, 3-ketovaleryl-CoA, acetyl- and propionyl-CoA and the corresponding acylphosphates, valeryl-CoA, and possibly 3-pentenoyl-CoA. With exception of the enzyme presumably reducing 2,4-pentadienoyl-CoA to 3-pentenoyl-CoA, enzymes catalyzing the formation and utilization of the above intermediates were demonstrated in extracts. Trans-2-pentenoyl-CoA was shown to be the immediate precursor of valeryl-CoA. The reduction of 2-pentenoyl-CoA was found to be coupled to the oxidation of 4-pentenoyl-CoA to 2,4-pentadienoyl-CoA. Several enzymes catalyzing the above reactions were partially purified and some of their properties determined. A high pressure liquid chromatography method of identifying and estimating most of the above mentioned CoA thiolesters was developed.

p-Cresol formation by cell-free extracts of Clostridium difficile.

Cell-free extracts of Clostridium difficile were shown to form p-cresol by decarboxylation of p-hydroxyphenylacetic acid. This activity required both high and low molecular weight fractions. The active component of the low molecular weight fraction had properties of an amino acid and could be replaced by serine, threonine or the corresponding alpha keto acids. Pyruvate was shown to function catalytically. Since the high molecular weight fraction was O2-sensitive and since dithionite was as effective as pyruvate with some high molecular weight fractions, the alpha keto acids probably serve as low potential reducing agents in this system. Because of instability, the p-cresol-forming enzyme could not be purified.

Pathway of lysine degradation in Fusobacterium nucleatum.

Lysine was fermented by Fusobacterium nucleatum ATCC 25586 with the formation of about 1 mol each of acetate and butyrate. By the use of [1-14C]lysine or [6-14C]lysine, acetate and butyrate were shown to be derived from both ends of lysine, with acetate being formed preferentially from carbon atoms 1 and 2 and butyrate being formed preferentially from carbon atoms 3 to 6. This indicates that the lysine carbon chain is cleaved between both carbon atoms 2 and 3 and carbon atoms 4 and 5, with the former predominating [1-14C]acetate was also extensively incorporated into butyrate, preferentially into carbon atoms 3 and 4. Cell-free extracts of F. nucleatum were shown to catalyze the reactions of the 3-keto,5-aminohexanoate pathway of lysine degradation, previously described in lysine-fermenting clostridia. The 3-keto,5-aminohexanoate cleavage enzyme was partially purified and shown to have properties much like those of the clostridial enzyme. We conclude that both the pathway and the enzymes of lysine degradation are similar in F. nucleatum and lysine-fermenting clostridia.

Metabolism of L-beta-lysine by a Pseudomonas. Purification and properties of a deacetylase-thiolesterase utilizing 4-acetamidobutyryl CoA and related compounds.

A deacetylase-thiolesterase that cleaves both the amide and thiolester bonds of 4-acetamidobutyryl CoA has been highly purified from extracts of Pseudomonas B4 grown in a medium containing L-beta-lysine (3,6-diaminohexanoate) as the main energy source. The enzyme has a molecular weight of about 275,000 and contains 8 apparently identical subunits of 36,500 daltons. Products of 4-acetamidobutyryl CoA degradation are stoichiometric amounts of CoASH and acetate, variable amounts of 4-aminobutyrate and its lactam, 2-pyrrolidinone, and a little 4-acetamidobutyrate. The relative yields of 4-aminobutyrate and 2-pyrrolidinone are determined by the enzyme level. At high enzyme levels the 4-aminobutyrate/pyrrolidinone ratio is about 2, whereas at low enzyme levels only pyrrolidinone is formed. Under the latter conditions, 4-aminobutyryl CoA accumulates transiently and is converted nonenzymatically to pyrrolidinone and CoASH. Since the enzyme does not form 4-aminobutyrate from synthetic or enzymatically formed 4-aminobutyryl CoA, we conclude that a 4-aminobutyryl CoA-enzyme complex is the actual precursor of 4-aminobutyrate, whereas free 4-aminobutyryl CoA is the precursor of pyrrolidinone. Several analogs of 4-acetamidobutyryl CoA containing different amino acid or amide moieties, and several simple acyl CoA compounds are utilized by the enzyme; 4-propionamidobutyryl CoA and 5-acetamidovaleryl CoA are most readily decomposed. Acetyl CoA is a very poor substrate. 3-Acetamidopropionyl CoA is first converted to acetate and beta-alanyl CoA and the latter compound is slowly hydrolyzed to beta-alanine and CoASH. Little deacetylase-thiolesterase is formed by bacteria grown in absence of beta-lysine, but another thiolesterase, lacking deacetylase activity, is produced. The deacetylase-thiolesterase catalyzes an essential step in the aerobic degradation of L-beta-lysine.

Enzymes involved in 3,5-diaminohexanoate degradation by Brevibacterium sp.

Cell-free extracts of Brevibacterium sp. L5 grown on DL-erythro-3,5-diaminohexanoate were found to contain a 3-keto-5-aminohexanoate cleavage enzyme that converts 3-keto-5-aminohexanoate and acetyl-coenzyme A (CokA) to 3-aminobutyryl-CoA and acetoacetate and a deaminase that coverts L-3-aminobutyryl-CoA to crotonyl-CoA. The cleavage enzyme has been purified extensively, and some of its properties have been determined for comparison with the 3-keto-6-acetamido-hexanoate cleavage enzyme of Pseudomonas sp. B4. The deaminase has been partially purified and characterized. Both the cleavage enzyme and the deaminase are induced by growth on 3,5-diaminohexanoate. The presence of these and other accessory enzymes in Brevibacterium sp. extracts accounts for the results of earlier tracer experiments which showed that C-1 and C-2 of 3-keto-5-aminohexanoate are converted mainly to acetoacetate and acetate, whereas C-3 to C-6 are converted mainly to 3-hydroxybutyrate or its coenzyme A thiolester. The enzymes observed in extracts of Brevibacterium sp. can account for the conversion of 3,5-diaminohexanoate to acetyl-CoA.

Purification and properties of 3-keto-5-aminohexanoate cleavage enzyme from a lysine-fermenting Clostridium.

The lysine-fermenting Clostridium SB4 is shown to contain a new type of beta-keto acid-degrading enzyme that converts 3-keto-5-aminohexanoate and acetyl-CoA reversibly to L-3-aminobutyryl-CoA and acetoacetate. Following the development of a sensitive radiochemical assay the enzyme was purified 175-fold to about 90% homogeneity in 44% yield. The specific activity of the purified enzyme is 44 IU/mg of protein at 30 degrees. The equilibrium constant for the forward reaction was found to be 0.68 at 30 degrees and pH 7.0, corresponding to a deltaG0' of 0.23 kcal/mol. The enzyme is highly substrate-specific. Of several substrate analogs tested in the forward and back reactions only beta-alanyl-CoA and D-3-aminobutyryl-CoA are utilized about 130% and 1.7% as fast as L-3-aminobutyryl-CoA, respectively. The product formed from beta-alanyl-CoA and acetoacetate is a neutral beta-keto acid, presumably 3-keto-5-aminopentanoic acid; its borohydride reduction product was partially characterized as a hydroxy-amino acid by various chromatographic and ion exchange methods. The activity of the purified enzyme is increased about 5-fold by addition of 0.1 mM Co2+ and to a lesser extent by Mn2+. Activity is inhibited by orthophosphate, thiol reagents, and EDTA; however, exposure of the enzyme to the latter compound prior to addition of Co2+ increases activity, presumably by removing competing divalent cations. Tracer experiments have shown that carbon atoms 1 and 2 of acetoacetate are derived from carbon atoms 1 and 2 of 3-keto-5-aminohexanoate whereas carbon atoms 3 and 4 are derived from acetyl-CoA. The amino acid moiety of 3-aminobutyryl-CoA is derived from carbon atoms 3 to 6 of 3-keto-5-aminohexanoate. Since no evidence for covalent enzyme-substrate intermediates could be obtained by the study of four possible group exchange reactions, a concerted reaction between 3-keto-5-aminohexanoate and acetyl-CoA is considered. The enzyme has a molecular weight of about 97,000 and probably contains four identical subunits. The relatively high specific activity of the enzyme in extracts of Clostridium SB4 indicates it functions in the main pathway of lysine degradation. This relatively stable enzyme provides a convenient and specific method for the quantitative estimation of nanomolar amounts of L- and D-3-aminobutyryl-CoA and beta-alanyl-CoA.