Mechanistic studies on the pyridoxal phosphate enzyme 1-aminocyclopropane-1-carboxylate deaminase from Pseudomonas sp

Abstract

The enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACPC deaminase) from a pseudomonad is a pyridoxal phosphate (PLP) linked catalyst which fragments the cyclopropane substrate to alpha-ketobutyrate and ammonia [Honma, M., & Shimomura, T. (1978) Agric. Biol. Chem. 42, 1825]. Enzymatic incubations in D2O yield alpha-ketobutyrate with one deuterium at the C-4 methyl group and one deuterium at one of the C-3 prochiral methylene hydrogens. Stereochemical analysis of the location of the C-3 deuteron was accomplished by in situ enzymatic reduction to (2S)-2-hydroxybutyrate with L-lactate dehydrogenase and conversion to the phenacyl ester. The C-3 hydrogens of the (2S)-2-hydroxybutyryl moiety are fully resolved in a 250-MHz NMR spectrum. Absolute assignment of 3S and 3R loci was obtained with phenacyl (2S,3S)-2-hydroxy[3-2H]butyrate generated enzymatically by D-serine dehydratase action on D-threonine. ACPC deaminase shows a stereoselective outcome with a 3R:3S deuterated product ratio of 72:28. 2-Vinyl-ACPC is also a fragmentation substrate with exclusive regiospecific cleavage to yield the straight-chain keto acid product 2-keto-5-hexenoate. The D isomer of vinylglycine is processed to alpha-ketobutyrate and ammonia at 8% the Vmax of ACPC, while L-vinylglycine is not a substrate. It is likely that ACPC and D-vinylglycine yield a common intermediate--the vinylglycine-PLP-p-quinoid adduct--which is then protonated sequentially at C-4 and then C-3 to account for the observed deuterium incorporation. The D isomers of beta-substituted alanines (fluoroalanine, chloroalanine, and O-acetyl-D-serine) partition between catalytic elimination and enzyme inactivation. Each shows a different partition ratio, arguing against the common aminoacrylyl-PLP as the inactivating species.

Substitutions of Alanine for Cysteine at a Reactive Thiol Site and for Lysine at a Pyridoxal Phosphate Binding Site of 1-Aminocyclopropane-1-carboxylate Deaminase

“…1,3) 2) one of the C-3 methylene hydrogens of the product, Ct-ketobutyrate, was exchanged with solvent hydrogen during the enzyme reaction. 4 ) Two alternative mechanisms consistent with these findings were discussed in a previous paper (Fig. 2).…”

A New 1-Aminocyclopropane-1-Carboxylic Acid-Conjugating Activity in Tomato Fruit

“…In a Pseudomonas species and in the yeast Hansenula saturnus ACC also is oxidatively deaminated to a-ketobutyrate and ammonia in a pyridoxal phosphate-dependent reaction (Walsh et al, 1981;Honma, 1985). We describe the identification in tissue extracts of tomato fruit of a y-glutamytranspeptidase activity, which catalyzes, in a glutathione-dependent reaction, the formation of GACC, a new conjugate of ACC.…”

Isolation, sequence, and expression in Escherichia coli of the Pseudomonas sp. strain ACP gene encoding 1-aminocyclopropane-1-carboxylate deaminase

“…Pseudomonas ACC deaminase has an estimated molecular mass of 110 kDa and is composed of three identical subunits, each with a molecular mass estimated to be 36.5 kDa (7,8). ACC deaminase utilizes pyridoxal 5'-phosphate as a cofactor in catalyzing the cleavage of ACC to a-ketobutyrate and ammonia (7,22).ACC was originally identified as a natural product in the juices of several fruits (2) and is now regarded as a key intermediate in the biosynthesis of the plant hormone ethylene (23). Ethylene is biosynthesized in higher plants from methionine via S-adenosylmethionine and ACC (1, 24).…”

“…Pseudomonas ACC deaminase has an estimated molecular mass of 110 kDa and is composed of three identical subunits, each with a molecular mass estimated to be 36.5 kDa (7,8). ACC deaminase utilizes pyridoxal 5'-phosphate as a cofactor in catalyzing the cleavage of ACC to a-ketobutyrate and ammonia (7,22).…”

“…The rate-limiting step in ethylene biosynthesis is the production of ACC by a ring-closing displacement of methylthioadenosine from S-adenosylmethionine in a reaction catalyzed by ACC synthase (1). The reverse of this reaction can be viewed as analogous to the reaction catalyzed by ACC deaminase (22).…”