Crystal Structure of Quinohemoprotein Alcohol Dehydrogenase from Comamonas testosteroni

Abstract

Quinoprotein alcohol dehydrogenases are redox enzymes that participate in distinctive catabolic pathways that enable bacteria to grow on various alcohols as the sole source of carbon and energy. The x-ray structure of the quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni has been determined at 1.44 Å resolution. It comprises two domains. The N-terminal domain has a ␤-propeller fold and binds one pyrroloquinoline quinone cofactor and one calcium ion in the active site. A tetrahydrofuran-2-carboxylic acid molecule is present in the substrate-binding cleft. The position of this oxidation product provides valuable information on the amino acid residues involved in the reaction mechanism and their function. The C-terminal domain is an ␣-helical type I cytochrome c with His 608 and Met 647 as heme-iron ligands. This is the first reported structure of an electron transfer system between a quinoprotein alcohol dehydrogenase and cytochrome c. The shortest distance between pyrroloquinoline quinone and heme c is 12.9 Å, one of the longest physiological edge-to-edge distances yet determined between two redox centers. A highly unusual disulfide bond between two adjacent cysteines bridges the redox centers. It appears essential for electron transfer. A water channel delineates a possible pathway for proton transfer from the active site to the solvent.Bacteria have versatile metabolic pathways that enable them to adapt to different environmental conditions. Many Gram-negative bacteria, for example, can grow on compounds as different as methylamine, ethanol, and glucose as their sole source of carbon and energy (1-3). The crucial, first step in the catabolism of such compounds is often an oxidation reaction catalyzed by a class of periplasmic enzymes called quinoproteins. Quinoproteins are oxidoreductases that possess one of four different quinone compounds instead of nicotinamide or flavin cofactors (4 -7). They oxidize a wide variety of alcoholand amine-containing substrates to the corresponding aldehydes or ketones. Proteins containing the pyrroloquinoline quinone (PQQ) 1 cofactor form the best characterized and largest quinoprotein subclass (8). Two different types of PQQ-containing alcohol dehydrogenases (ADHs) have been characterized. The first type includes quinoprotein ethanol dehydrogenases (EDHs) from several Pseudomonas species (9 -11) and quinoprotein methanol dehydrogenases (MDHs) from methylotrophic bacteria (12). The second type of PQQ-dependent ADHs is the quinohemoprotein alcohol dehydrogenases (QH-ADHs). In addition to PQQ, these latter enzymes contain a covalently bound heme c. Both soluble monomeric QH-ADHs (2, 11, 13-16) and membrane-associated enzymes that consist of several subunits (17-19) have been described.No three-dimensional structures of QH-ADHs are known. In contrast, several x-ray structures have been reported of type I quinoprotein ADHs (20 -24) and a soluble quinoprotein glucose dehydrogenase (sGDH) (25). sGDH-inhibitor (26) and sGDHsubstrate (27) complexes have provided detail...

Structural Basis of Redox-coupled Protein Substrate Selection by the Cytochrome c Biosynthesis Protein ResA

“…Many bacteria also contain enzymes with heme c and at least one other redox cofactor, e.g. hemocytochromes c (4), flavocytochromes c (5, 6), and quinocytochromes c (7). Recent findings that mono-heme c-type cytochromes function as apoptosis-triggering factors in eukaryotes (8) and as sensors of the toxic signaling molecule nitric oxide (9) have sparked new interest in these proteins.…”

Novel Dehalogenase Mechanism for 2,3-Dichloro-1-Propanol Utilization in Pseudomonas putida Strain MC4

“…The complete dppA-encoded protein sequence, including the signal peptide (698 amino acids [aa]), was very similar to type II quinohemoprotein alcohol dehydrogenases, particularly with the 2-chloroethanol dehydrogenase from Pseudomonas stutzeri (78% identity), the homologous alcohol dehydrogenases IIB (76%) and IIG (53%) from P. putida HK5 (48, 49), a type I quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni (51%) (39,45), and a tetrahydrofurfuryl alcohol dehydrogenase from Ralstonia eutropha Bo (51% identity) (58). Homology with less than 50% identity was found with a quinoprotein ethanol dehydrogenase from P. aeruginosa (38% identity) (13, 31) and a methanol dehydrogenase from Methylophilus methylotrophus W3A1 (35% identity) (56).…”

Cloning and expression of the gene for periplasmic poly(vinyl alcohol) dehydrogenase from Sphingomonas sp. strain 113P3, a novel-type quinohaemoprotein alcohol dehydrogenase

“…Activity of PVADH from strain VM15C toward PPGs has not been confirmed. The structure and function of type II QH-ADHs was well characterized by X-ray crystallography with ADH from C. testosteroni (Oubrie et al, 2002) and ADH IIB from P. putida HK5 (Chen et al, 2002). Type II QH-PVADHs have a structural motif common to this group, a quinoprotein-specific superbarrel domain (see the sequence alignment available as supplementary data with the online version of this paper), where PQQ is deeply embedded in the centre, and a unique haem-binding c domain.…”