Crystallographic Evidence for Substrate-assisted Catalysis in a Bacterial β-Hexosaminidase

Abstract

Carbohydrates are involved in many diverse biological functions including cell structural integrity, energy storage, pathogen defense and invasion mechanisms, viral penetration, and cellular signaling. Therefore, a large number of enzymes dedicated to carbohydrate metabolism have evolved. Enzymes specifically responsible for carbohydrate catabolism are collectively referred to as glycosyl hydrolases and have been classified into 77 families based on amino acid sequence similarity (1-3). Three-dimensional structures are known for representatives of 30 of the families. Although there are differences in chain length and domain structure between proteins of a single family, all proteins of a family hydrolyze the glycosidic bond with the same stereochemical outcome (4).Family 20 includes the ␤-N-acetylhexosaminidases (␤-hexosaminidases) 1 (EC 3.2.1.52), enzymes that catalyze the removal of terminal ␤-1,4 linked N-acetylhexosamine residues from the nonreducing ends of oligosaccharides and their conjugates. In humans, there are two major ␤-hexosaminidase isoforms: HexA and HexB. HexA is a heterodimer of subunits ␣ (encoded by HEXA) and ␤ (encoded by HEXB), whereas HexB is a homodimer of ␤ subunits. HexA is essential for degrading GalNAc-␤(1,4)-[N-acetylneuraminic acid (2,3)]-Gal-␤(1,4)-Glcceramide ganglioside; the biological importance of HexA activity is illustrated by the fatal neurodegenerative disorders that result from its heritable deficiency (5). Mutations in HEXA or HEXB cause Tay-Sachs and Sandhoff disease, respectively. These genetic diseases have made the human ␤-hexosaminidase isoforms the subject of much research. A substantial amount of genetic and biochemical information is available for these isozymes (5), but detailed information about their catalytic mechanism is limited. Mechanistic studies have been primarily limited by the difficulties in producing sufficient amounts of recombinant enzyme needed for kinetic analysis (6, 7); however, recent improvements in expression and purification procedures have allowed more accurate kinetic measurements to be made (8). Crystals of human HexB have been grown (9); however, attempts at solving its three-dimensional structure have not been successful. Nonetheless, much insight into the mechanism of human HexA and HexB has been provided by structural and functional studies carried out on related family 20 glycosyl hydrolases (10 -12).Stereochemical outcome studies on the family 20 chitobiase from Serratia marcescens (13) and human ␤-hexosaminidase (14) demonstrated that this family operates via a retaining * This work was supported in part by the

Kinetic evidence related to substrate‐assisted catalysis of family 18 chitinases

“…In family 18 chitinases, the proton donor residue has been shown to be a glutamate, whereas the residue that acts as the nucleophile has not been detected by either mutational or X-ray diffraction analyses [2][3][4], indicating that the reaction mechanism of family 18 chitinases differs from that of other retaining enzymes. Similar situations have also been observed with family 20 glycosidases (e.g., b-N-acetylhexosaminidase) and family 56 glycosidases (e.g., hyaluronidase) [5][6][7][8].…”

“…This is a strong support for the substrate assisted catalysis mechanism, because compound 4 was designed to reduce the polarization of the carbonyl (thionyl) group in order to weaken the nucleophilic effect of the oxygen (sulfur) atom. Furthermore, the 3D-structure of a family-20 b-N-acetylhexosaminidase from Streptomyces plicatus complexed with GlcNAc-thiazoline (8) suggested that the enzymatic hydrolysis proceeded via a cyclic intermediate [5,6].…”

Crystal Structure of β-Hexosaminidase B in Complex with Pyrimethamine, a Potential Pharmacological Chaperone

“…27 In order to locate the PYR ligand, a difference Fourier map was calculated with the Collaborative Computational Project 4 (CCP4) 28 suite of programs (S Fall , 29 Sigma-A, 30 and Refmac5 31 for rigid body refinement) using the PDB coordinates (1NOU) of the native β-hexosaminidase B. 1 PYR, sugars, SO 4 ions, and water molecules were not included in the initial rigid body refinement and map calculations but were added in subsequent rounds. Further rounds of refinement were also carried out with Refmac5.…”

“…All the members of this family use a substrate-assisted catalysis mechanism, which is achieved by positioning the C2-acetamido group of the terminal GalNAc into the appropriate position for nucleophilic attack of the carbonyl oxygen atom on C1. 4,5 The substrate binding environment also ensures that a β-configuration is retained by directing the position of the incoming nucleophilic water molecule. A detailed description of the catalytic mechanism can be found in the paper by Lemieux et al 6 Genetic defects in either gene encoding the subunits of HexA can result in the accumulation of G M2 ganglioside in neural tissues and two of three lysosomal storage diseases collectively known as G M2 gangliosidosis.…”

Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

“…Although GH3 enzymes, including NagZ, are functionally related to the GH20 human b-hexosaminidase isoenzymes and GH84 O-GlcNAcase, recent kinetic [17][18][19] and structural studies 16,[20][21][22] have revealed that GH3 enzymes use a catalytic mechanism that differs from that used by GH20 and GH84 enzymes. This distinction should therefore offer a tractable route to generating selective inhibitors of NagZ, and clear comparisons of these enzymes at a structural level would greatly accelerate these efforts.…”