Structural and Functional Characterization of YdjI, an Aldolase of Unknown Specificity in Escherichia coli K12

Abstract

The ydj gene cluster is found in 80% of sequenced Escherichia coli genomes and other closely related species in the human microbiome. On the basis of the annotations of the enzymes located in this cluster, it is expected that together they catalyze the catabolism of an unknown carbohydrate. The focus of this investigation is on YdjI, which is in the ydj gene cluster of E. coli K-12. It is predicted to be a class II aldolase of unknown function. Here we describe a structural and functional characterization of this enzyme. YdjI catalyzes the hydrogen/deuterium exchange of the pro-S hydrogen at C3 of dihydroxyacetone phosphate (DHAP). In the presence of DHAP, YdjI catalyzes an aldol condensation with a variety of aldo sugars. YdjI shows a strong preference for higher-order (seven-, eight-, and nine-carbon) monosaccharides with specific hydroxyl stereochemistries and a negatively charged terminus (carboxylate or phosphate). The best substrate is L-arabinuronic acid with an apparent k cat of 3.0 s −1 . The product, L-glycero-L-galacto-octuluronate-1-phosphate, has a k cat /K m value of 2.1 × 10 3 M −1 s −1 in the retro-aldol reaction with YdjI. This is the first recorded synthesis of L-glycero-L-galacto-octuluronate-1-phosphate and six similar carbohydrates. The crystal structure of YdjI, determined to a nominal resolution of 1.75 Å (Protein Data Bank entry 6OFU), reveals unusual positions for two arginine residues located near the active site. Computational docking was utilized to distinguish preferable binding orientations for L-glycero-L-galacto-octuluronate-1-phosphate. These results indicate a possible alternative binding orientation for L-glycero-L-galacto-octuluronate-1-phosphate compared to that observed in other class II aldolases, which utilize shorter carbohydrate molecules.

Deciphering the Aldolase Function of STM3780 from a Bovine Enteric Infection-Related Gene Cluster in Salmonella enterica Serotype Typhimurium

“…The 13 C NMR spectrum of the single reaction product was compared with the NMR spectrum of authentic D-xylulonate-1-P, synthesized enzymatically by the reaction catalyzed by YdjI. 10 The 13 C NMR spectrum was identical (Figure 4). The product of the reaction was further dephosphorylated and compared with the 13 C NMR spectrum previously reported for D-xylulonate (also known as 4-keto-D-arabonate) and shown to be the same.…”

“…Therefore, STM3780 catalyzes the abstraction of the same prochiral hydrogen as rabbit FBP aldolase, which is the pro-S hydrogen at C3 of DHAP. 10 Product Stereochemistry at C4. To determine the stereochemistry at C4 of the product in the condensation reaction, STM3780 was mixed with DHAP and glyoxylate 1 to generate either D-xylulonate-1-P or L-ribulonate-1-P as illustrated in Scheme 1.…”

“…We have previously shown that this compound is also a substrate for YdjI aldolase from E. coli K12, also giving L-galacto-heptuluronate-1-P 9 as the reaction product after condensation with DHAP. 10 However, the physiological significance of this observation is uncertain at this point. Surprisingly, DHAP was shown to undergo a selfcondensation reaction catalyzed by STM3780 to form dendroketose bisphosphate.…”

“…A similar reaction mixture was made using YdjI as the catalyst for the synthesis of D-xyluonate-1-P from DHAP and glyoxylate. 10 Purification of STM3780 Reaction Products. Selected products of the STM3780-catalyzed reactions were purified so they could be further characterized.…”

“…For those having DHAP as a product, they can be grouped into two clusters of orthologous groups, COG0191 and COG0235, where they require a different stereochemical arrangement at C3 . COG0191 includes the functionally annotated aldolases, fructose-bisphosphate aldolase (FbaA), d -tagatose bisphosphate aldolase (KbaY and GatY), 2-deoxy-5-keto- d -gluconate-6-phosphate aldolase (IolJ), and fructose-1,6-bisphosphate aldolase homologue (YdjI), all of which require an S stereocenter at C3, and COG0235 includes l -rhamnulose-1-phosphate aldolase (RhaD), l -fuculose-1-phosphate aldolase (FucA), and 3-oxo-tetronate 4-phosphate decarboxylase (OtnC or YgbL) requiring an R stereocenter at C3. − Recently, YdjI from E. coli was shown to prefer higher-order monosaccharides with the best substrate identified to be l - glycero - l - galacto -octulonate 1-phosphate . STM3780 is currently annotated as a putative class II fructose-bisphosphate aldolase found in COG0191.…”