Role of Glu312 in Binding and Positioning of the Substrate for the Hydride Transfer Reaction in Choline Oxidase^,

Abstract

Choline oxidase catalyzes the oxidation of choline to glycine betaine, a compatible solute that accumulates in pathogenic bacteria and plants so they can withstand osmotic and temperature stresses. The crystal structure of choline oxidase was determined and refined to a resolution of 1.86 A with data collected at 100 K using synchrotron X-ray radiation. The structure reveals a covalent linkage between His99 Nepsilon2 and FAD C8M atoms, and a 123 A3 solvent-excluded cavity adjacent to the re face of the flavin. A hypothetical model for choline docked into the cavity suggests that several aromatic residues and Glu312 may orient the cationic substrate for efficient catalysis. The role of the negative charge on Glu312 was investigated by engineering variant enzymes in which Glu312 was replaced with alanine, glutamine, or aspartate. The Glu312Ala enzyme was inactive. The Glu312Gln enzyme exhibited a Kd value for choline at least 500 times larger than that of the wild-type enzyme. The Glu312Asp enzyme had a kcat/KO2 value similar to that of the wild-type enzyme but kcat and kcat/Km values that were 230 and 35 times lower, respectively, than in the wild-type enzyme. These data are consistent with the spatial location of the negative charge on residue 312 being important for the oxidation of the alcohol substrate. Solvent viscosity and substrate kinetic isotope effects suggest the presence of an internal equilibrium in the Glu312Asp enzyme prior to the hydride transfer reaction. Altogether, the crystallographic and mechanistic data suggest that Glu312 is important for binding and positioning of the substrate in the active site of choline oxidase.

Amino Acid Residues Critical for the Specificity for Betaine Aldehyde of the Plant ALDH10 Isoenzyme Involved in the Synthesis of Glycine Betaine

“…The positive charge of the quaternary nitrogen of the trimethylammonium group of BAL suggests that negatively charged active-site residues should be involved in conferring substrate specificity to plant BADHs, by analogy with other enzymes that bind this group (Quaye et al, 2008). In a first study with SoBADH, Glu-103, which is strictly conserved in the known plant ALDH10 enzymes, was thought to be this residue; when mutated to Gln, however, there were no changes in the kinetics with BAL as substrate and only a small negative effect on those with APAL and ABAL (Incharoensakdi et al, 2000).…”

Formate Oxidase, an Enzyme of the Glucose-Methanol-Choline Oxidoreductase Family, Has a His-Arg Pair and 8-Formyl-FAD at the Catalytic Site

“…5,6,[9][10][11] FOD AO was crystallized in space group C222 1 (Table 1), in which three subunits are contained in the asymmetric unit. FOD AO has been found to be a dimeric protein in solution by size-exclusion chromatography and SDS-PAGE.…”

“…Their locations are similar to those of Arg 87 and Ser 94 respectively. [9][10][11] A potential interaction between Arg 87 and the formyl group or that between Ser 94 and the formyl group might be important to the stabilization of 8-formyl-FAD in FOD AO .…”

“…Although they exhibit low amino acid sequence similarities at their catalytic sites, 1) the crystal structures of the enzymes, such as cholesterol oxidase, [2][3][4] glucose oxidase, 5,6) pyranose 2-oxidase, 7,8) choline oxidase, [9][10][11] and arylalcohol oxidase, 12) show that all of them share a highly conserved catalytic site. In the vicinity of the pyrimidine part of the isoalloxazine ring, a His residue conserved through this family of enzymes pairs with an Asn residue or a His residue.…”

Biochemical Analysis of Recombinant AlkJ from Pseudomonas putida Reveals a Membrane-Associated, Flavin Adenine Dinucleotide-Dependent Dehydrogenase Suitable for the Biosynthetic Production of Aliphatic Aldehydes

“…In fact, attempts to dissociate AlkJ from the bacterial membrane with high concentrations of NaCl, a procedure commonly used for disrupting the electrostatic interaction of peripheral membrane proteins, remained unsuccessful (not shown). This behavior distinguishes AlkJ from CHOX, the closest homologue with known X-ray structure (37). Although AlkJ and CHOX are both grouped within the class of GMC oxidoreductases and have a similar fold, the subcellular localizations of the two enzymes appear to be markedly different, since the latter was described as a soluble protein (43).…”