Longyin Chen

Crystallographic investigations of the tryptophan-derived cofactor in the quinoprotein methylamine dehydrogenase

A model of tryptophan tryptophylquinone (TTQ), recently proposed by Mclntire et al. (Science (1991) 252, 817‐824) to be the prosthetic group of the quinoprotein methylamine dehydrogenase, has been compared with electron density maps of this dehydrogenase from Thiobacillus versutus and Paracoccus denitrificans. The comparison shows that the TTQ model can be neatly accommodated, providing strong supportive evidence that TTQ is indeed the cofactor for this group of quinoproteins.

Preliminary X-ray crystallographic studies of methylamine dehydrogenase and methylamine dehydrogenase-amicyanin complexes from Paracoccus denitrificans

Single crystals of methylamine dehydrogenase and of complexes between methylamine dehydrogenase and both amicyanin and apo-amicyanin from Paracoccus denitrificans have been prepared by the vapour diffusion method. The methylamine dehydrogenase crystals belong to the orthorhombic space group P212121 and have unit cell parameters a = 151·78 A, b = 135·84 A and c = 55·14 A. The two complex crystals are isomorphous and are tetragonal, space group P41212 or its enantiomer, and have cell parameters a = b = 124·6 A and c = 247·3 A. The crystals diffract to beyond 2·8 A resolution.

Structural studies of methylamine dehydrogenase

Methylamine dehydrogenase (MADH), found in methyldtrophic bacteria, is an inducible, periplasmic quinoenzyme which catalyzes the oxidation of methylamine to formaldehyde and ammonia (De Beer R et al. 1980). It contains the novel cofactor tryptophan tryptophylquinone (TTQ), which is derived from two tryptophan side chains (McIntire WS et al. 1991). Subsequently, electrons are transferred to the membrane bound terminal oxidase, cytochrome aa3 via a series of soluble electron carrier proteins. In facultative autotrophs, such as Paracoccus denitrificans, the initial electron acceptor in this chain is amicyanin, a blue copper protein (Husain M, Davidson VL 1985), while in Methylophilus methylotrophus W3A1, which lacks the gene for amicyanin (Chistoserdov AY et al. 1994) the initial acceptor is a cytochrome c552 (Chandrasekar R Clapper MH 1986). In the case of P. denitrificans, in vitro studies suggest that the acceptor following amicyanin in the electron transfer chain is cytochrome c551i(Husain M, Davidson VL 1986). All three proteins are induced when these bacteria are grown on methylamine as the sole carbon source.

The ternary complex between methylamine dehydrogenase, amicyanin and cytochrome c551i

The structure of a ternary complex between methylamine dehydrogenase (MADH), amicyanin and cytochrome c551i, all from Paracoccus denitrificans has been determined at 2.4 A resolution. MADH and amicyanin associate so that the exposed edges of Tip 108 of TTQ and the His 95 ligand of copper are juxtaposed. Amicyanin and cytochrome c551i associate so that one edge of the β-sandwich of amicyanin is in contact with a chain segment of the cytochrome close to the heme propionates. The distance from the catalytically active quinone oxygen of TTQ to the copper is 16.8 A and from the copper to the iron is 24.8 A, respectively. Two efficient paths for electron flow from TTQ to copper were found, one passing through Tip 108 of MADH. Two paths from copper to iron were also found, one through the cysteine and one through the methionine ligand to copper, which converge at Tyr 30 of amicyanin.