Crystal structure of 1-aminocyclopropane-1-carboxylate deaminase from Hansenula saturnus.

The pyridoxal 5'-phosphate (PLP)-dependent enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD) catalyzes a reaction that involves a ring opening of cyclopropanoid amino acid, yielding alpha-ketobutyrate and ammonia. Unlike other PLP-dependent enzymes, this enzyme has no alpha-hydrogen atom in the substrate. Thus, a unique mechanism for the bond cleavage is expected. The crystal structure of ACCD from Hansenula saturnus has been determined at 2.0 A resolution by the multiple wavelength anomalous diffraction method using mercury atoms as anomalous scatterers. The model was built on the electron density map, which was obtained by the density averaging of multiple crystal forms. The final model was refined to an R-factor of 22.5% and an R(free)-factor of 26.8%. The ACCD folds into two domains, each of which has an open twisted alpha/beta structure similar to the beta-subunit of tryptophan synthase. However, in ACCD, unlike in other members of the beta family of PLP-dependent enzymes, PLP is buried deep in the molecule. The structure provides the first view of the catalytic center of the cyclopropane ring opening.

Properties, sequence, and synthesis in Escherichia coli of 1-aminocyclopropane-1-carboxylate deaminase from Hansenula saturnus.

The plant hormone ethylene is generated from a unique precursor, 1-aminocyclopropane-1-carboxylate (ACC). In previous studies, ACC deaminase, which degrades ACC to alpha-ketobutyrate and ammonia, was found in four strains of Pseudomonas, characterized, and sequenced. To verify the wider distribution of ACC deaminase in microorganisms, we purified and sequenced ACC deaminase from the yeast Hansenula saturnus. The purified enzyme was active toward ACC, D-serine and dl-coronamic acid, indicating the same stereospecificity as the Pseudomonas enzyme, but unlike the bacterial enzyme it was not active toward beta-chloro-D-alanine and O-acetyl-D-serine. Analyses of peptides from proteolytic digests of the purified and modified ACC deaminase covered more than 90% of its amino acid sequence and showed a blocked N-terminal residue as N-acetylserine. A cDNA encoding the ACC deaminase was isolated from H. saturnus cells incubated in alpha-aminoisobutyrate medium, and sequenced. The yeast enzyme has 441 amino acid residues, of which 60 to 63% are identical to those of reported Pseudomonas enzymes. The open reading frame encoding ACC deaminase was subcloned into pET-11d and expressed in Escherichia coli BL21 (DE3) as an active enzyme.