Fermentation of D-α-lysine. Purification and properties of an adenosine triphosphate regulated B12-coenzyme-dependent D-α-lysine mutase complex from Clostridium sticklandii

Abstract

The enzyme system from Clostridium sticklandii that catalyzes the migration of the 6-amino group of D-alysine to carbon 5, forming 2,5-diaminohexanoate, has been purified to near homogeneity as a complex of two dissimilar proteins, a red cobamide protein (Ei) and a sulfhydryl protein (E2). The complex is dissociated by acidification to pH 4.0; the precipitate which forms contains Ei while E2 remains in the supernatant solution. The combined fractions are catalytically active. The activity of the isolated D-a-lysine mutase complex depends on added pyridoxal phosphate, Bi2-coenzyme, and a mercaptan. Mutase activity is stimulated by ATP, Mg2+, and a monovalent cation such as K+ or NH4+. ATP, which can be replaced by its phosphonic acid analogs, is an allon the basis of early isotope studies it was concluded that the fermentation of DL-lysine to acetate, butyrate, and ammonia by Clostridium sticklandii involves two separate pathways (Stadtman and White, 1954;. In one pathway, which could be investigated in soluble extracts (Stadtman, 1963), acetate is formed from carbon atoms 1 and 2 of lysine and butyrate is formed from the remainder of the molecule. The primary substrate, L-a-lysine, first undergoes two successive amino group migration reactions (Chirpich et al., 1970;Tsai and Stadtman, 1968;Stadtman and Renz, 1968) and then is oxidatively deaminated to form 3-keto-5aminohexanoate (Rimerman and Barker, 1968) before eventual cleavage of the carbon chain occurs (Figure 1).In the other pathway acetate is formed from carbons 5 and 6 of lysine and butyrate is derived from the carboxyl end of the molecule. Soluble extracts fail to carry out this overall series of reactions and, therefore, the reaction sequence is still unknown. However, the recently discovered conversion of D-a-lysine into 2,5-diaminohexanoate (Stadtman and Tsai, 1967) presumably is the first step. (Figure 1). In both lysine pathways there is a Bi2-coenzyme-mediated migration of the e-amino group to carbon 5; in the L-lysine pathway this is the second enzymic step and the product is 3,5-diaminohexanoate whereas in the D-lysine pathway it is the first step and the product is 2,5-diaminohexanoate. The two Bi2-coenzyme-• From the

Regulation of CFTR Cl- channel gating by ADP and ATP analogues.

“…While there is wide variation in the Po subsequent to ATP addition in the presence of ATP analogues, the data clearly demonstrate that a threefold lower concentration of ATP is able to stimulate and support a 6-to 12-fold increase in channel Po compared to the channel activity that was seen in the presence of the ATP analogue alone. This is a clear divergence from other systems in which ATP analogues have been shown to be effective on an equimolar basis at mimicking the effects of ATP (Morley and Stadtman, 1970;Yount, et al, 1971b;Hayden, Miller, Brauweiler, and Bamburg, 1993). Subsequent experiments were designed to determine ifATP analogues altered the effect of ATP on CFTR chloride channels.…”

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

“…One such pathway that operates in several bacterial species is the fermentation of lysine to yield acetate. Interestingly, the lysine fermentation pathway contains two analogous enzymes: lysine 5,6-aminomutase (5,6-LAM), which is AdoCbl-dependent (6,7), and lysine 2,3-aminomutase (2,3-LAM), which is an S-adenosylmethionine (AdoMet or SAM)-dependent ironsulfur enzyme (8)(9)(10). Both enzymes require pyridoxal 5Ј-phosphate (PLP) (8,11) in addition to AdoCbl or AdoMet, and both catalyze a 1,2 amino group shift with concomitant H atom migration (Fig.…”

Lysine Metabolism by Clostridia

“…The characteristic colored products formed when the various diaminohexanoates are heated with the acid ninhydrin reagent of Chinard (28) have been exploited both for the qualitative identification and quantitative estimation (12,17,29) of these amino acids.…”

“…In both of the lysine fermentation pathways the primary attack on the substrate involves migration of one or both of the amino groups. In addition to the resulting diamino acid intermediates (10)(11)(12)(13)(14), all of the products of the numbered reactions and the enzymes responsible for their formation have been identified in both C. sticklandii and Clostridium strain M-E (5,12,(15)(16)(17)(18)(19)(20). The enzymes that catalyze reactions 1,3,4, and 5 also have been identified in a third lysine-fermenting organism, Clostridium strain (13,(22)(23)(24)(25)(26).…”