Identification of biochemical and putative biological role of a xenolog from Escherichia coli using structural analysis

Abstract

YagE is a 33 kDa prophage protein encoded by CP4-6 prophage element in Escherichia coli K12 genome. Here, we report the structures of YagE complexes with pyruvate (PDB Id 3N2X) and KDGal (2-keto-3-deoxy galactonate) (PDB Id 3NEV) at 2.2A resolution. Pyruvate depletion assay in presence of glyceraldehyde shows that YagE catalyses the aldol condensation of pyruvate and glyceraldehyde. Our results indicate that the biochemical function of YagE is that of a 2-keto-3-deoxy gluconate (KDG) aldolase. Interestingly, E. coli K12 genome lacks an intrinsic KDG aldolase. Moreover, the over-expression of YagE increases cell viability in the presence of certain bactericidal antibiotics, indicating a putative biological role of YagE as a prophage encoded virulence factor enabling the survival of bacteria in the presence of certain antibiotics. The analysis implies a possible mechanism of antibiotic resistance conferred by the over-expression of prophage encoded YagE to E. coli.

Biochemical and Structural Characterization of l-2-Keto-3-deoxyarabinonate Dehydratase: A Unique Catalytic Mechanism in the Class I Aldolase Protein Superfamily

“…The secondary structural elements for AbAraD are shown above the alignment, and the colors correspond to Figure B. Highlighted active sites of AbAraD complexed with 2-oxobutyrate (B), and its superimposition with KdgD from A. tumefaciens (complex with 2-oxobutyrate) (C), KdgD from Oceanobacillus iheyensis (ligand-free) (D), HypD from S. meliloti (ligand-free) (E), the hypothetical protein from R. palustris (ligand-free) (F), KdgA from S. solfataricus [complex with d -2-keto-3-deoxygalactonate (KDGal)] (G), YagE from E. coli (complex with KDGal) (H), LRA4 from S. stipitis (complex with KDGal) (I), Hog1 from humans (ligand-free) (J), and PhdJ from M. vanbaalenii [complex with trans - o -carboxybenzylidenepyruvate (CBP)] (K). The colors of active site residues shown as sticks with carbon atoms correspond to those in panel A. Double-headed arrows with bond lengths indicate the distance between the side chain carboxyl oxygen of Glu171 in AbAraD and the side chain hydroxyl of the conserved tyrosine in other DHDPS/NAL members.…”

“…In the "E 2 αβ mechanism" (Figure 6A), the reaction may start when the neutral side chain of Lys171 makes a nucleophilic attack on the carbonyl C2 atom of the substrate, leading to the formation of imine intermediate 1, a Schiff base. Deprotonation of the C3 atom by Glu173 as the (first) base catalyst may 23 (C), KdgD from Oceanobacillus iheyensis (ligand-free) 51 (D), HypD from S. meliloti (ligand-free) 43 (E), the hypothetical protein from R. palustris (ligand-free) (F), KdgA from S. solfataricus [complex with D-2-keto-3-deoxygalactonate (KDGal)] 19 (G), YagE from E. coli (complex with KDGal) 24 (H), LRA4 from S. stipitis (complex with KDGal) (I), Hog1 from humans (ligand-free) 42 Although no candidate assisted in the expulsion of the O4 hydroxyl by acting as a (first) acid catalyst, the main chain carbonyl of Met216 was located in the vicinity of the βhydroxyl of hydroxypyruvate (Figure 4E), which was structurally equivalent to the "side chain" hydroxyl of Ser211 in KdgD from A. tumefaciens (Figure 5A,C), and the "main chain" carbonyl of Ile203 in DHDPS from E. coli. The alanine mutant of Ser211 of KdgD exhibited markedly reduced activity.…”

“…This enzyme belonged to the DHDPS/NAL protein superfamily, including the archetypical dihydrodipicolinate synthase (DHDPS) and N -acetylneuraminate lyase (NAL) (Figure ). Among them, aldolases for 2-keto-3-deoxysugar acids were involved in the route I pathways of d -glucose (KdgA , ), l -fucose (FucH), l -rhamnose (LRA4), d -galacturonate (Lga1 , ), and d -xylonate metabolism (YagE) from archaea, fungi, and bacteria (Figure A–E). All DHDPS/NAL members possessed a common (β/α) 8 barrel fold and shared a common catalytic mechanism for retro-aldol fission, in which a lysine residue formed a Schiff base with the carbonyl C2 atom of the substrate, followed by proton extraction of the substrate by a tyrosine residue as a catalytic base (Figure S1B).…”

Engineering microbial pathways for production of bio-based chemicals from lignocellulosic sugars: current status and perspectives

“…2 ). In E. coli , two genes yjhH and yagE encode a functional KDPA [ 62 ], as evidenced by the finding that a double yjhH yagE mutant was unable to grow on d -xylonate [ 63 ]. In addition, KDPA encoded by yagE is likely more active than that encoded by yjhH since levels of GA were higher in engineered strains with yagE [ 63 ].…”

Characterization of l-2-keto-3-deoxyfuconate aldolases in a nonphosphorylating l-fucose metabolism pathway in anaerobic bacteria

“…6) and functions as D-KDP aldolase in D-xylonate metabolism (Fig. 1, E and G) (11,19). Therefore, LRA4, LGA1, and YagE from Pichia stipites (also named Scheffersomyces stipitis), Hypocrea jecorina (also named Trichoderma reesei), and Escherichia coli were enzymatically characterized in detail using a library of 2-keto-3-deoxysugar acids (referred to as PsLRA4, HjLGA1, and EcYagE, respectively).…”