Structure determination of quinoprotein methylamine dehydrogenase from Thiobacillus versutus

Abstract

The crystal structure of quinoprotein methylamine dehydrogenase from Thiobacillus versutus (EC 1.4.99.3, Mr = 123 500) has been solved to 2-25 ,~ resolution. The crystals of space group P3~21 (a = b = 129-8, c = 104.3A) contain half a tetrameric enzyme molecule in the asymmetric unit, with a solvent content of ca 70%. The procedure used to solve this structure involved multiple isomorphousreplacement phasing, complemented by phase extension using solvent flattening, and phase combination with partial-model phases. The use of solvent flattening was essential to generate good quality electron density maps into which initial models were built. These partial models were refined using molecular-0108-7681/90/060806-18503.00 dynamics procedures. Refined model phases were then combined with solvent-flattening phases to generate improved electron density distributions. In the absence of an amino-acid sequence for this enzyme, the current 2-25 A resolution electron density map was interpreted to provide a model for the complete molecule. The crystallographic R factor for this model, which lacks any water molecules, is 28.6% for data between 6.0 and 2.25 ,~ resolution.

The catalytic mechanism of glyceraldehyde 3-phosphate dehydrogenase from Trypanosoma cruzi elucidated via the QM/MM approach

“…23 reported a new location for the site Pi in the GAPDH of Trypanosoma cruzi, where the phosphate group of a G3P analogue forms hydrogen bonds with residues Thr226 and Arg249. In this conformation, the phosphonate group is 3.35 Å to the position previously described for the site of the phosphate in GAPDH L. mexicana 26 and 4.81 Å to the position described in Trypanosoma brucei (Vellieux, Hajdu, and Hol, 1995). 27 28 reported a new location for the Pi site in GAPDH in T. cruzi.…”

“…In this conformation, the phosphonate group is 3.35 Å to the position previously described for the site of the phosphate in GAPDH L. mexicana 26 and 4.81 Å to the position described in Trypanosoma brucei (Vellieux, Hajdu, and Hol, 1995). 27 28 reported a new location for the Pi site in GAPDH in T. cruzi. This new binding site is very close to that previously identified in the 3D structure of GAPDH in T. cruzi reported by .…”

“…The crystal structures of GAPDH have been determined at different resolution from various organisms, among them American lobster, 15,49 humans, 41,42 Bacillus stearothermophilus, 18,22,50 Thermotoga maritima, 25 Thermus aquaticus, 43 Escherichia coli, 44 Leishmania mexicana, 26,45 Trypanosoma brucei 27,46 and T. cruzi. 23,47,48 In this study, the initial coordinates for computational simulations were taken from the tetrameric crystal structure of the T. cruzi GAPDH complex at 1.95 Å resolution (PDB code 1K3T).…”

N‐terminal heterogeneity of methylamine dehydrogenase from Thiobacillus versutus

“…With difference of only three additional residues at the Nterminus of fragment c2, the sequence of cl was the same as that of the 32 kDa fragment that had been found earlier and that has been used to proof the backbone tracing of the polypeptide chain in the X-ray structure [9]. From that latter analysis it had appeared that the 'crystallographic' N-terminus of the a subunit was 90 residues upstream the 32 kDa fragment sequence.…”

“…At that momemt, it became clear from the X-ray data available [9], that the sequence of fragment c2 could be identified in the backbone pattern of MADH. With difference of only three additional residues at the Nterminus of fragment c2, the sequence of cl was the same as that of the 32 kDa fragment that had been found earlier and that has been used to proof the backbone tracing of the polypeptide chain in the X-ray structure [9].…”