Crystal Structure of 1-Aminocyclopropane-1-carboxylate Deaminase from Hansenula saturnus

Min Yao,T Ose,Hiroshi Sugimoto,Atsushi Horiuchi,Atsushi Nakagawa,Soichi Wakatsuki,Daisuke Yokoi,Toyotaka Murakami,Mamoru Honma,Isao Tanaka

doi:10.1074/jbc.m004681200

Abstract

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.

Highlights

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 ␣-ketobutyrate and ammonia
The crystal structure of ACCD from Hansenula saturnus has been determined at 2.0 Å resolution by the multiple wavelength anomalous diffraction method using mercury atoms as anomalous scatterers
Structure Description—Native yACCD was crystallized in the orthorhombic form of the space group C2221 with unit cell dimensions of a ϭ 65.7 Å, b ϭ 268.5 Å, and c ϭ 187.2 Å

Summary

EXPERIMENTAL PROCEDURES

Crystallization and Data Collection—The yACCD was purified and crystallized as described previously [11, 13]. For the mercury derivative of the trigonal form, MAD data were collected at the BL18B station of Photo Factory, Tsukuba, Japan, with a Weissenberg camera [14] using two different wavelengths (1.0090 Å (edge) and 0.9800 Å (peak)), which were chosen at the L-III edge. Because neither mercury derivative was isomorphous with the native crystals, the “remote data” and “peak data” were each treated as “native” in the orthorhombic and trigonal forms, respectively. The initial electron density map was obtained after phase improvement by the program SOLOMON [20] implemented in SHARP [19]. After electron density averaging of multiple crystal forms using the program RAVE [21] combined with SOLOMON [20] and the CCP4 program package [16], the atomic model was built using the baton and lego in the O program [23].

RESULTS AND DISCUSSION

Ra Cullis

TABLE III The final refinement statistics