Abstract
Various monovalent cations influence the enzymatic activity and the spectroscopic properties of methylamine dehydrogenase (MADH). Here, we report the structure determination of this tryptophan tryptophylquinone-containing enzyme from Methylobacterium extorquens AM1 by high resolution x-ray crystallography (1.75 A). This first MADH crystal structure at low ionic strength is compared with the high resolution structure of the related MADH from Paracoccus denitrificans recently reported. We also describe the first structures (at 1.95 to 2.15 A resolution) of an MADH in the substrate-reduced form and in the presence of trimethylamine and of cesium, two competitive inhibitors. Polarized absorption microspectrophotometry was performed on single crystals under various redox, pH, and salt conditions. The results show that the enzyme is catalytically active in the crystal and that the cations cause the same spectral perturbations as are observed in solution. These studies lead us to propose a model for the entrance and binding of the substrate in the active site.
Highlights
Various monovalent cations influence the enzymatic activity and the spectroscopic properties of methylamine dehydrogenase (MADH)
The first class comprises the MADH from Methylobacterium extorquens AM1 (MADH-AM) [4], Thiobacillus versutus (MADH-TV) [5], and Paracoccus denitrificans (MADH-PD) [6] that interact with the cupredoxin amicyanin to form a transient electron transfer complex [7]
The resolution of the native MADH-AM structure, 1.75 Å, is comparable with that of MADH-PD [11]. The structures of these two forms of the enzyme are remarkably similar, even to the extent of maintaining the same three residues of the heavy subunit in a strained conformation, Ile-128(H), His-209(H), and cis-Pro184(H). This similarity exists despite the differences in sequence identity for the H and L subunits (32% and 13%) and the differences in ionic strength during crystallization (ϳ3 M ammonium sulfate versus ϳ20% PEG4000)
Summary
Vol 273, No 40, Issue of October 2, pp. 25703–25712, 1998 Printed in U.S.A. Crystallographic and Spectroscopic Studies of Native, Aminoquinol, and Monovalent Cation-bound Forms of Methylamine Dehydrogenase from Methylobacterium extorquens AM1*. We report the structure determination of this tryptophan tryptophylquinone-containing enzyme from Methylobacterium extorquens AM1 by high resolution x-ray crystallography (1.75 Å). The results show that the enzyme is catalytically active in the crystal and that the cations cause the same spectral perturbations as are observed in solution These studies lead us to propose a model for the entrance and binding of the substrate in the active site. Methylamine dehydrogenase (MADH) (EC 1.4.99.3) catalyzes the oxidation of methylamine in the periplasm of various methylotrophic bacteria to provide the organism with carbon, nitrogen, and energy [1]. This enzyme is a quinoprotein and contains the novel cofactor tryptophan tryptophylquinone. The reaction catalyzed by MADH is as follows: CH3NH3ϩ ϩ H2O 3 NH4ϩ ϩ HCHO ϩ 2Hϩ ϩ 2eϪ
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