Crystal structures of d-psicose 3-epimerase from Clostridium cellulolyticum H10 and its complex with ketohexose sugars

Abstract

D-psicose 3-epimerase (DPEase) is demonstrated to be useful in the bioproduction of D-psicose, a rare hexose sugar, from D-fructose, found plenty in nature. Clostridium cellulolyticum H10 has recently been identified as a DPEase that can epimerize D-fructose to yield D-psicose with a much higher conversion rate when compared with the conventionally used DTEase. In this study, the crystal structure of the C. cellulolyticum DPEase was determined. The enzyme assembles into a tetramer and each subunit shows a (β/α)(8) TIM barrel fold with a Mn(2+) metal ion in the active site. Additional crystal structures of the enzyme in complex with substrates/products (D-psicose, D-fructose, D-tagatose and D-sorbose) were also determined. From the complex structures of C. cellulolyticum DPEase with D-psicose and D-fructose, the enzyme has much more interactions with D-psicose than D-fructose by forming more hydrogen bonds between the substrate and the active site residues. Accordingly, based on these ketohexose-bound complex structures, a C3-O3 proton-exchange mechanism for the conversion between D-psicose and D-fructose is proposed here. These results provide a clear idea for the deprotonation/protonation roles of E150 and E244 in catalysis.

Recent advances in d -allulose: Physiological functionalities, applications, and biological production

“…At last, the Glu152 protonated C-3, and the two Glu residues reached the negatively ionized state (Yoshida et al, 2007). In 2012, Chan et al successfully crystallized both substrate-bound and product-bound complex structures of C. cellulolyticum DPEase, which gave more evidence of the C3eO3 proton-exchange mechanism and offered a clear method for the deprotonation/protonation effects of Glu150 and Glu 244 in the isomerization process (Chan et al, 2012).…”

Advances in the enzymatic production of l-hexoses

“…The enzyme was first immobilized on Chitopearl beads BCW 2510, and the immobilized enzyme produced 20 % L-tagatose and 65 % L-fructose from 40 g L −1 of Lsorbose and 20 g L −1 of L-psicose, respectively (Itoh and Izumori 1996). The crystal structure of ketose 3-epimerases from P. cichorii (PDB: 2QUL) (Yoshida et al 2007), A. tumefaciens (PDB: 2HK0) (Kim et al 2006b), C. cellulolyticum (PDB: 3VNI) (Chan et al 2012), and M. loti (PDB: 3VYL) (Uechi et al 2013a) has been determined and is available in the Protein Data Bank. Based on already known crystal structure information, a molecular modification made to these epimerases would be expected to improve the substrate specificity and enzymatic activity toward L-ketohexoses, and thus, the enzymatic production of L-ketohexoses could be more efficient and widely utilized in the near future.…”

Structure ofD-tagatose 3-epimerase-like protein fromMethanocaldococcus jannaschii

“…This enzyme is thus expected to be key to the mass production of d-psicose from d-fructose (Takeshita et al, 2000). To date, the crystal structures of d-TE from P. cichorii (Yoshida et al, 2007), d-psicose 3-epimerases (d-PEs) from Agrobacterium tumefaciens (Kim et al, 2006) and Clostridium cellulolyticum (Chan et al, 2012), and l-ribulose 3-epimerase (l-RE) from Mesorhizobium loti (Uechi et al, 2013) have all been solved. Extensive analysis of these structures has provided detailed information about the substratebinding sites and has led to elucidation of the substrate-recognition and catalytic mechanisms of the enzymes.…”

“…2a). When this model of the MJ1311p monomer was sent to the DALI server (Holm & Rosenströ m, 2010) Chan et al, 2012). As shown in Fig.…”

“…It has been reported that P. cichorii d-TE exhibits a dimeric structure (Yoshida et al, 2007), whereas C. cellulolyticum and A. tumefaciens dPEs exhibit tetrameric structures (Chan et al, 2012;Kim et al, 2006). The arrangement of the four subunits of the latter two enzymes is essentially the same.…”