Structure and function of Caulobacter crescentus aldose–aldose oxidoreductase

Abstract

Aldose-aldose oxidoreductase (Cc AAOR) is a recently characterized enzyme from the bacterial strain Caulobacter crescentus CB15 belonging to the glucose-fructose oxidoreductase/inositol dehydrogenase/rhizopine catabolism protein (Gfo/Idh/MocA) family. Cc AAOR catalyses the oxidation and reduction of a panel of aldose monosaccharides using a tightly bound NADP(H) cofactor that is regenerated in the catalytic cycle. Furthermore, Cc AAOR can also oxidize 1,4-linked oligosaccharides. In the present study, we present novel crystal structures of the dimeric Cc AAOR in complex with the cofactor and glycerol, D-xylose, D-glucose, maltotriose and D-sorbitol determined to resolutions of 2.0, 1.8, 1.7, 1.9 and 1.8 Å (1 Å=0.1 nm), respectively. These complex structures allowed for a detailed analysis of the ligand-binding interactions. The structures showed that the C1 carbon of a substrate, which is either reduced or oxidized, is close to the reactive C4 carbon of the nicotinamide ring of NADP(H). In addition, the O1 hydroxy group of the substrate, which is either protonated or deprotonated, is unexpectedly close to both Lys(104) and Tyr(189), which may both act as a proton donor or acceptor. This led us to hypothesize that this intriguing feature could be beneficial for Cc AAOR to catalyse the reduction of a linear form of a monosaccharide substrate and the oxidation of a pyranose form of the same substrate in a reaction cycle, during which the bound cofactor is regenerated.

On the structure and function of Escherichia coli YjhC: An oxidoreductase involved in bacterial sialic acid metabolism

“…Within the Gfo/Idh/MocA family, members who modify carbohydrates typically catalyze their respective reactions via a ping-pong mechanism, where the cofactor binds first, followed by the substrate. 17,18,51 In the absence of NAD + / NADH, the carbon compounds tested gave only small thermal shifts, within a range of 0.06 C to 0.37 C, which we did not consider significant (Table S1). However, in the presence of NAD + /NADH, both Neu5Ac and the sialic acid 1,7 lactone variant had a significant effect on the thermal stability of YjhC with an additional thermal shift of 2.86 C and 0.98 C, respectively ( Figure 2C,D).…”

Structural insights into the catalytic and substrate recognition mechanisms of bacterial l‐arabinose 1‐dehydrogenase

“…The crystal structure of AraDH was determined using the molecular replacement method with the crystal structure of R. etli D-galactose 1-dehydrogenase (PDB code, 4EW6; 56% sequence identity with AraDH) as a search model and subsequently refined to 1.5 A resolution against the dataset from a crystal of AraDH (Fig. Structural comparison between AraDH and other members of the Gfo/Idh/ MocA family; Zymomonas mobilis glucose-fructose oxidoreductase (ZmGFOR; PDB code, 1OFG; 20% sequence identity with AraDH) [23] and Caulobacter crescentus aldose-aldose oxidoreductase (CcAAOR; PDB code, 5A03; 20% sequence identity with AraDH) [24] shows the r.m.s. The asymmetric unit of the crystal contains two AraDH molecules, which have a similar conformation to each other, with root mean square (r.m.s.)…”

“…1B). Structural comparison between AraDH and other members of the Gfo/Idh/ MocA family; Zymomonas mobilis glucose-fructose oxidoreductase (ZmGFOR; PDB code, 1OFG; 20% sequence identity with AraDH) [23] and Caulobacter crescentus aldose-aldose oxidoreductase (CcAAOR; PDB code, 5A03; 20% sequence identity with AraDH) [24] shows the r.m.s. difference of 1.9 and 1.9…”

Structural and functional features of the NAD(P) dependent Gfo/Idh/MocA protein family oxidoreductases

“…The final analysis contained 11 different proteins: 1) G6PD from Leuconostoc mesenteroides, 1 2) GFOR from Zymomonas mobilis, ketoreductase (KijD10) from Actinomadura kijaniata, 9 and 11) the novel AAOR from Caulobacter crescentus. 4 The structures are listed in Table I with their PDB IDs, EC numbers (concluded from the catalyzed reaction or published in the article related to the structure), catalytic activity, number of amino acid residues, quaternary structures, utilized cofactors, and the calculated root-mean square deviations (RMSD) superimposing the secondary structures to Cc AAOR. The sequence identities between the proteins were generally low, under 20%, in spite of many common structural features, such as a NAD(P) binding site.…”

“…Especially AAOR catalyzes also the reactions of a panel of different sugars. 4,5 GP6D is more specific, it catalyses oxidation (dehydrogenation) of D-glucose-6-phosphate to 6-phospho-D-glucono-1,5-lactone. 1 AFR reduces C2 carbon of 1,5-anhydro-Dfructose, the central intermediate of the anhydrofructose pathway.…”