Contribution of Flavin Covalent Linkage with Histidine 99 to the Reaction Catalyzed by Choline Oxidase

Abstract

A number of enzymes, including dehydrogenases (1-3), monooxygenases (4 -7), halogenases (8 -11), and oxidases (7, 12, 13), employ flavin cofactors (FAD or FMN) for their catalytic processes. About a tenth of all flavoproteins have been shown to contain a covalently attached cofactor, which may be linked at the C8M position via histidyl, tyrosyl, or cysteinyl side chains or at the C6M position via a cysteinyl side chain (14). Glucooligosaccharide oxidase (15, 16), hexose oxidase (17), and berberine bridge enzyme (18,19) The discovery of quantum mechanical tunneling in enzymatic reactions, in which hydrogen atoms, protons, and hydride ions are transferred, has attracted considerable interest in enzyme studies geared toward understanding the mechanisms underlying the several orders of magnitudes in the rate enhancements of protein-catalyzed reactions compared with non-enzymatic ones. Tunneling mechanisms have been shown in a wide array of cofactor-dependent enzymes, including flavoenzymes. Examples of flavoenzymes in which the tunneling mechanisms have been demonstrated include morphinone reductase (29, 30), pentaerythritol tetranitrate reductase (29), glucose oxidase (31-33), and choline oxidase (34). Mechanistic data on Class 2 dihydroorotate dehydrogenases, also with a flavin cofactor (FMN) covalently linked to the protein moiety (35,36), could only propose a mechanism that is either stepwise or concerted with significant quantum mechanical tunneling for the hydride transfer from C6 and the deprotonation at C5 in the oxidation of dihydroorotate to orotate (37). This leaves choline oxidase as the only characterized enzyme with a covalently attached flavin cofactor (12,38), where the oxidation of its substrate occurs unequivocally by quantum mechanical tunneling.Choline oxidase from Arthrobacter globiformis catalyzes the two-step FAD-dependent oxidation of the primary alcohol substrate choline to glycine betaine with betaine aldehyde, which is predominantly bound to the enzyme and forms a gem-diol species, as intermediate (Scheme 1). Glycine betaine accumulates in the cytoplasm of plants and bacteria as a defensive mechanism against stress conditions, thus making genetic engineering of relevant plants of economic interest (39 -45), and the biosynthetic pathway for the osmolyte is a potential drug target in human microbial infections of clinical interest (46 -48). The first oxidation step catalyzed by choline oxidase involves the transfer of a hydride ion from a deprotonated choline to the protein-bound flavin followed by reaction of the anionic flavin hydroquinone with molecular oxygen to regenerate the oxidized FAD (for a recent review see Ref. 50). The gem-diol choline, i.e. hydrated betaine aldehyde, is the substrate for the second oxidation step (49), suggesting that the reaction may follow a similar mechanism. The isoalloxazine ring of the flavin cofac-

Genetic Control of Osmoadaptive Glycine Betaine Synthesis in Bacillus subtilis through the Choline-Sensing and Glycine Betaine-Responsive GbsR Repressor

“…Microorganisms can synthesize glycine betaine by one of two different routes: (i) through a stepwise methylation of the amino acid glycine (39,46,59) or (ii) through the import and subsequent oxidation of choline using various combinations of enzymes (5,15,42,49,51). B. subtilis uses this latter route to produce glycine betaine and imports for this purpose the precursor choline into the cell via two osmotically inducible ABC-type transport systems: the aforementioned OpuC transporter and the OpuB system ( Fig.…”

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

“…18,19) The choline and glucose oxidases have covalently bound FAD in an 8-N "2 -histydyl linkage and non-covalently bound FAD respectively. [20][21][22] In the choline and glucose oxidases, the His 466 -Asn 510 and His 516 -His 559 pairs respectively are situated close to the pyrimidine part of the isoalloxazine ring, respectively. 5,6,9) Comparisons of the amino acid sequences of the FODs with those of the choline and glucose oxidases suggest that the His 511 -Arg 554 pair and the His 508 -Arg 551 pair are situated close to the pyrimidine part of the isoalloxazine ring in recombinant Aspergillus oryzae FOD (FOD AO ) and the Debaryomyces FODs respectively.…”

Pathway of Glycine Betaine Biosynthesis in Aspergillus fumigatus

“…The percentage of identity between the choline oxidases of A. fumigatus and Arthrobacter globiformis, which is the only bacterial species that has a unique CHOA gene, was 37% (37). In addition, the amino acids in the catalytic site of the A. globiformis choline oxidase are conserved in A. fumigatus and other fungi, as follows: H99, site of flavin covalent attachment (38); H310, proton relay with H466; E312, substrate binding (39); H351, hydrogen bonding OH of choline (40); V464, substrate positioning and oxygen reactivity (41); H466, positive charge stabilization of the alkoxide intermediate of the reaction (42); S101, facilitating hydride transfer (43); and N510, involved in the reductive and oxidative half reaction (44). Both the fungal and bacterial enzymes display steady-state kinetic mechanisms with formation of a ternary complex with either choline or BA as the substrate, with oxygen reacting with the reduced flavin before the organic product of the reaction is released from the enzyme active site (37).…”