Coenzyme B6 dependent novel bond cleavage reactions

Coenzyme B6 is an important cofactor participating in many diverse biotransformations. W e the function of this coenzyme in enzyme catalysis has been well established, recent studies have led to two novel coenzyme B6-dependent reactions whose catalyses involve unique mechanistic variants of usual coenzyme B6 chemistry. These are a C 0 bond cleavage and a C-C bond cleavage reaction catalyzed by CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (El) and 1aminocyclopropane1-carboxylate deaminase (ACC deaminase), respectively. Enzyme El is the only known pyridoxamine 5'-phosphate (PMP) dependent ironsulfur containing dehydratase, and it is one of a few &dependent enzymes with a radical mechanism. ACC deaminase is a pyridoxal 5'-phosphate (PLP) dependent enzyme which acts on a cyclopropane substrate, and catalyzes the cleavage between the Ca-Cp bond. Current knowledge concerning the properties and mechanisms of these unusual enzymes is summarized in this paper. As a coenzyme, vitamin B6 phosphate exhibits a remarkably versatile array of catalytic roles, ranging from transaminations, racemization, pand y-eliminatiodsubstitution, decarboxylation, etc (1). The achievement of these catalyses by B6-dependent enzymes involves a common recurring theme, relying on the electron-withdrawing capability of this cofactor to stabilize the anion generated at the a-C of the substratecoenzyme complex. The transiently delocalized electrons are later used for the cleavage and/or formation of covalent bonds. While extensive knowledge about the genetics, structure, function, and mechanism of this class of enzymes have been accumulated over the years, several recent studies have revealed a few new traits of coenzyme B6 that are beyond the scope previously established for its catalyzed reactions. For example, the traditional role of coenzyme B6 has been varied by lysine-2,3-aminomutase which utilizes PLP to catalyze a radical rearrangement of the a-amino group of lysine (2). A Bg-dependent aminotransferase was also implicated in the conversion of cytosylglucuronic acid to blasticidin S (3). The role of this as yet to be isolated enzyme in blasticidin biosynthesis has been hypothesized not only to act as a transaminase but also to catalyze the dideoxygenation at C-2' and C-3' of the glucuronate residue. In this paper, two other unusual cases of coenzyme B6 dependent reactions will be presented: one involved in C-3 deoxygenation of a 4-keto6-deoxyhexose and the other in the ring opening of a cyclopropane amino acid, both of which have been the subjects of our research in recent years. The current information regarding their modes of action will be summarized herein. CDP-6-deoxy-L-threo-D-gZycero-4-hexulose-3-dehydrase (El) Enzyme El is a homo-dimeric protein (49 KDa per subunit) requiring a pyridoxamine 5'-phosphate (PMP) and an adrenodoxidputidaredoxin-type [2Fe-2S] center per monomer for full activity (4). This enzyme, together with its reductase (CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase, E3). plays an essential role in C 0 bond severance in the biosynthesis of 3,6-dideoxyhexoses (5). Specifically, El and E3 catalyze the conversion of CDP-6-deoxy-L-threo-D-glycero-4-hexulose (1) to the c 3 deoxygenated product, CDP-3,6-dideoxy-D-glycero-D-glycero-4-hexulose (2). As depicted in Scheme 1, the mechanism of El catalysis has been established to be initiated by the formation of a Schiff base (3) between PMP and the C-4 keto group of the substrate (44, followed by the abstraction of the p r o 4 proton from the C-4' position of the resulting adduct 3 (4b) to trigger the expulsion of the C-3 hydroxyl group leading to a A3*4-glUCOSet?n intermediate 4 in the active site of El. While intermediate 4 has never been isolated, the reversible nature of its hydration was demonstrated by isotopic incorporation with [l*O]H20 (4a). Furthermore, replacement of the 3-OH group by a solvent hydrogen in the E1-E3 coupled reaction has been observed to proceed with retention of configuration (7). In sum, these results imply that the overall catalysis in the active site of El is likely a suprafacial process occurring on the si face of the PMP-substrate complex (4). as is the case with most other coenzyme B6-dependent enzymes (1). In fact, sequence align-