Β-Glucuronidase Explained

β-Glucuronidases are members of the glycosidase family of enzymes that catalyze breakdown of complex carbohydrates.^[1] Human β-glucuronidase is a type of glucuronidase (a member of glycosidase Family 2) that catalyzes hydrolysis of β-D-glucuronic acid residues from the non-reducing end of mucopolysaccharides (also referred to as glycosaminoglycans) such as heparan sulfate.^{[1] [2] [3]} Human β-glucuronidase is located in the lysosome.^[4] In the gut, brush border β-glucuronidase converts conjugated bilirubin to the unconjugated form for reabsorption. β-Glucuronidase is also present in breast milk, which contributes to neonatal jaundice. The protein is encoded by the GUSB gene in humans^{[5] [6]} and by the uidA gene in bacteria.^[7]

Structure

Human β-glucuronidase is synthesized as an 80 kDa monomer (653 amino acids) before proteolysis removes 18 amino acids from the C-terminal end to form a 78 kDa monomer.^{[8] [9]} β-Glucuronidase exists as a 332 kDa homotetramer.^[10] β-Glucuronidase contains several notable structural formations, including a type of β-barrel known as a jelly roll barrel and a TIM barrel.

Mechanism of catalysis

Human β-glucuronidase is homologous to the Escherichia coli enzyme β-galactosidase.^{[11] [12]} This homologous relationship, along with the knowledge that glycosidases often perform hydrolysis catalyzed by two acidic residues, enabled the development of a mechanistic hypothesis. This hypothesis proposes that the two glutamic acid residues Glu540 and Glu451 are the nucleophilic and acidic residues, respectively, and that the tyrosine residue Tyr504 is also involved in catalysis. In support of this hypothesis, experimental mutations in any of these three residues result in large decreases of enzymatic activity. Increased activity of an E451A mutant enzyme (where Glu451 is replaced with an alanine residue) after addition of azide is consistent with Glu451 as the acid/base residue.^[13] Using analysis of labeled β-glucuronidase peptides after hydrolysis of a substrate that enters a very stable intermediate stage, researchers have determined that Glu540 is the nucleophilic residue.^[14]

Though the particular type of nucleophilic substitution employed by β-glucuronidase is unclear, evidence for the mechanisms of their homologues in the glycosidase family suggests that these reactions are qualitatively S_N2 reactions. The reactions proceed through a transition state with oxocarbenium ion characteristics. Initially, these mechanisms, because of this oxocarbenium characteristic of the transition state, were suggested to be S_N1 reactions proceeding through a discrete oxocarbenium ion intermediate. However, more recent evidence suggests that these oxocarbenium ion states have lifetimes of 10 femtoseconds - 0.1 nanoseconds (similar to that of a bond vibration period). These lifetimes are too short to assign to a reaction intermediate. From this evidence, it appears that these reactions, while having an S_N1 appearance due to the oxocarbenium ion characteristics of their transition states, must be qualitatively S_N2 reactions.^[1]

The specific activity of Tyr504 in the catalytic mechanism is unclear.^[13] Through comparison to the structural data of the homologous enzyme xylanase, it has been suggested that Tyr504 of β-glucuronidase might stabilize the leaving nucleophile (Glu540) or modulate its activity.

In addition to these residues, a conserved asparagine residue (Asn450) has been suggested to stabilize the substrate through the action of a hydrogen bond at the 2-hydroxyl group of the sugar substrate.^[10]

Sly syndrome

See main article: Sly syndrome. Deficiencies in β-glucuronidase result in the autosomal recessive inherited metabolic disease known as Sly syndrome or Mucopolysaccharidosis VII. A deficiency in this enzyme results in the build-up of non-hydrolyzed mucopolysaccharides in the patient. This disease can be extremely debilitating for the patient or can result in hydrops fetalis prior to birth. In addition, mental retardation, short stature, coarse facial features, spinal abnormalities, and enlargement of liver and spleen are observed in surviving patients.^[4] This disease has been modeled in a strain of mice as well as a family of dogs.^{[15] [16]} More recently researchers have discovered a feline family that exhibits deficiencies in β-glucuronidase activity. The source of this reduction of activity has been identified as an E351K mutation (Glu351 is mutated to a lysine residue). Glu351 is conserved in mammalian species, which suggests an important function for this residue. Examination of the human X-ray crystal structure suggests that this residue (Glu352 in the human enzyme), which is buried deep within the TIM barrel domain, may be important for stabilization of the tertiary structure of the enzyme.^[17] In the crystal structure, it appears that Arg216, a member of the jelly roll domain of the protein, forms a salt bridge with Glu352; therefore, Glu352 is likely involved in stabilizing the interaction between two different three-dimensional domains of the enzyme.

Use as a reporter gene

In molecular biology, β-glucuronidase is used as a reporter gene to monitor gene expression in mammalian and plant cells. Monitoring β-glucuronidase activity through the use of a GUS assay allows determination of the spatial and temporal expression of the gene in question.^[18]

• Molecular graphics images were produced using the Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco.^[19]

See also

• Alpha-glucuronidase
• Glucuronosyl-disulfoglucosamine glucuronidase
• Glycyrrhizinate beta-glucuronidase

Further reading

• George J . Elevated serum beta-glucuronidase reflects hepatic lysosomal fragility following toxic liver injury in rats . Biochemistry and Cell Biology . 86 . 3 . 235–43 . June 2008 . 18523484 . 10.1139/O08-038 .
• Bell CE, Sly WS, Brot FE . Human beta-glucuronidase deficiency mucopolysaccharidosis: identification of cross-reactive antigen in cultured fibroblasts of deficient patients by enzyme immunoassay . The Journal of Clinical Investigation . 59 . 1 . 97–105 . January 1977 . 401508 . 333336 . 10.1172/JCI108627 .
• Tanaka J, Gasa S, Sakurada K, Miyazaki T, Kasai M, Makita A . Characterization of the subunits and sugar moiety of human placental and leukemic beta-glucuronidase . Biological Chemistry Hoppe-Seyler . 373 . 1 . 57–62 . January 1992 . 1311180 . 10.1515/bchm3.1992.373.1.57 .
• Wolfe JH, Sands MS, Barker JE, Gwynn B, Rowe LB, Vogler CA, Birkenmeier EH . 4337590 . Reversal of pathology in murine mucopolysaccharidosis type VII by somatic cell gene transfer . Nature . 360 . 6406 . 749–53 . 1993 . 1465145 . 10.1038/360749a0 .
• Tomatsu S, Fukuda S, Sukegawa K, Ikedo Y, Yamada S, Yamada Y, Sasaki T, Okamoto H, Kuwahara T, Yamaguchi S . Mucopolysaccharidosis type VII: characterization of mutations and molecular heterogeneity . American Journal of Human Genetics . 48 . 1 . 89–96 . January 1991 . 1702266 . 1682743 .
• Shipley JM, Miller RD, Wu BM, Grubb JH, Christensen SG, Kyle JW, Sly WS . Analysis of the 5' flanking region of the human beta-glucuronidase gene . Genomics . 10 . 4 . 1009–18 . August 1991 . 1916806 . 10.1016/0888-7543(91)90192-H .
• Ono M, Taniguchi N, Makita A, Fujita M, Sekiya C, Namiki M . Phosphorylation of beta-glucuronidases from human normal liver and hepatoma by cAMP-dependent protein kinase . The Journal of Biological Chemistry . 263 . 12 . 5884–9 . April 1988 . 10.1016/S0021-9258(18)60648-9 . 2833520 . free .
• Guise KS, Korneluk RG, Waye J, Lamhonwah AM, Quan F, Palmer R, Ganschow RE, Sly WS, Gravel RA . Isolation and expression in Escherichia coli of a cDNA clone encoding human beta-glucuronidase . Gene . 34 . 1 . 105–10 . 1985 . 3924735 . 10.1016/0378-1119(85)90300-2 .
• Ho YC, Ho LH, Ho KJ . Human hepatic beta-glucuronidase: an enzyme kinetic study . Enzyme . 33 . 1 . 9–17 . 1985 . 3987656 . 10.1159/000469398 .
• Shipley JM, Klinkenberg M, Wu BM, Bachinsky DR, Grubb JH, Sly WS . Mutational analysis of a patient with mucopolysaccharidosis type VII, and identification of pseudogenes . American Journal of Human Genetics . 52 . 3 . 517–26 . March 1993 . 7680524 . 1682147 .
• Vervoort R, Lissens W, Liebaers I . Molecular analysis of a patient with hydrops fetalis caused by beta-glucuronidase deficiency, and evidence for additional pseudogenes . Human Mutation . 2 . 6 . 443–5 . 1994 . 8111412 . 10.1002/humu.1380020604 . 46432543 . free .
• Wu BM, Sly WS . Mutational studies in a patient with the hydrops fetalis form of mucopolysaccharidosis type VII . Human Mutation . 2 . 6 . 446–57 . 1994 . 8111413 . 10.1002/humu.1380020605 . 21484555 . free .
• Maruyama K, Sugano S . Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides . Gene . 138 . 1–2 . 171–4 . January 1994 . 8125298 . 10.1016/0378-1119(94)90802-8 .
• Moullier P, Bohl D, Heard JM, Danos O . 26122567 . Correction of lysosomal storage in the liver and spleen of MPS VII mice by implantation of genetically modified skin fibroblasts . Nature Genetics . 4 . 2 . 154–9 . June 1993 . 8348154 . 10.1038/ng0693-154 .
• Shipley JM, Grubb JH, Sly WS . The role of glycosylation and phosphorylation in the expression of active human beta-glucuronidase . The Journal of Biological Chemistry . 268 . 16 . 12193–8 . June 1993 . 10.1016/S0021-9258(19)50325-8 . 8505339 . free .
• Nishimura Y, Kato K, Himeno M . Biochemical characterization of liver microsomal, Golgi, lysosomal, and serum beta-glucuronidases in dibutyl phosphate-treated rats . Journal of Biochemistry . 118 . 1 . 56–66 . July 1995 . 8537326 . 10.1093/oxfordjournals.jbchem.a124892 .
• Jain S, Drendel WB, Chen ZW, Mathews FS, Sly WS, Grubb JH . Structure of human beta-glucuronidase reveals candidate lysosomal targeting and active-site motifs . Nature Structural Biology . 3 . 4 . 375–81 . April 1996 . 8599764 . 10.1038/nsb0496-375 . 28862883 .
• Vervoort R, Islam MR, Sly WS, Zabot MT, Kleijer WJ, Chabas A, Fensom A, Young EP, Liebaers I, Lissens W . Molecular analysis of patients with beta-glucuronidase deficiency presenting as hydrops fetalis or as early mucopolysaccharidosis VII . American Journal of Human Genetics . 58 . 3 . 457–71 . March 1996 . 8644704 . 1914559 .
• Bonaldo MF, Lennon G, Soares MB . Normalization and subtraction: two approaches to facilitate gene discovery . Genome Research . 6 . 9 . 791–806 . September 1996 . 8889548 . 10.1101/gr.6.9.791 . free .
• Dentino AR, Raj PA, De Nardin E . Subtle differences between human and rabbit neutrophil receptors shown by the secretagogue activity of constrained formyl peptides . Archives of Biochemistry and Biophysics . 337 . 2 . 267–74 . January 1997 . 9016822 . 10.1006/abbi.1996.9791 .

External links

• • Updated research on reporter glucuronidase and other reporters from Reportergene
• Database of Catalytic Mechanism Research and other information on beta-glucuronidase

Notes and References

1. Book: Sinnott M . Comprehensive Biological Catalysis . 1 . Academic Press . 1998 . Manchester, UK . 119–138 . 978-0-12-646864-9 .
2. McCarter JD, Withers SG . Mechanisms of enzymatic glycoside hydrolysis . Current Opinion in Structural Biology . 4 . 6 . 885–92 . December 1994 . 7712292 . 10.1016/0959-440X(94)90271-2 .
3. 10.1021/cr00105a006 . Sinnott ML . Catalytic mechanisms of enzymic glycosyl transfer . Chem Rev . 90 . 7 . 1171–1202 . 1990.
4. Book: Nyhan . William L. . Bruce . Barshop . Pinar . Ozand . vanc . Atlas of Metabolic Diseases . 2 . Hodder Arnold . 2005 . London, UK . 501–503, 546–550 . 978-0-340-80970-9.
5. Oshima A, Kyle JW, Miller RD, Hoffmann JW, Powell PP, Grubb JH, Sly WS, Tropak M, Guise KS, Gravel RA . Cloning, sequencing, and expression of cDNA for human β-glucuronidase . Proceedings of the National Academy of Sciences of the United States of America . 84 . 3 . 685–9 . February 1987 . 3468507 . 304280 . 10.1073/pnas.84.3.685 . 1987PNAS...84..685O . free .
6. Web site: Entrez Gene: GUSB glucuronidase, beta.
7. Martins MT, Rivera IG, Clark DL, Stewart MH, Wolfe RL, Olson BH . Distribution of uidA gene sequences in Escherichia coli isolates in water sources and comparison with the expression of beta-glucuronidase activity in 4-methylumbelliferyl-β-D-glucuronide media . Applied and Environmental Microbiology . 59 . 7 . 2271–6 . July 1993 . 10.1128/AEM.59.7.2271-2276.1993 . 8357258 . 182268 . 1993ApEnM..59.2271M .
8. Islam MR, Grubb JH, Sly WS . C-terminal processing of human beta-glucuronidase. The propeptide is required for full expression of catalytic activity, intracellular retention, and proper phosphorylation . The Journal of Biological Chemistry . 268 . 30 . 22627–33 . October 1993 . 10.1016/S0021-9258(18)41574-8 . 8226771 . free .
9. Shipley JM, Grubb JH, Sly WS . The role of glycosylation and phosphorylation in the expression of active human β-glucuronidase . The Journal of Biological Chemistry . 268 . 16 . 12193–8 . June 1993 . 10.1016/S0021-9258(19)50325-8 . 8505339 . free .
10. Kim HW, Mino K, Ishikawa K . Crystallization and preliminary X-ray analysis of endoglucanase from Pyrococcus horikoshii . Acta Crystallographica. Section F, Structural Biology and Crystallization Communications . 64 . Pt 12 . 1169–71 . December 2008 . 19052378 . 2593689 . 10.1107/S1744309108036919 .
11. Henrissat B, Bairoch A . New families in the classification of glycosyl hydrolases based on amino acid sequence similarities . The Biochemical Journal . 293 (Pt 3) . 3 . 781–8 . August 1993 . 8352747 . 1134435 . 10.1042/bj2930781 .
12. Henrissat B . A classification of glycosyl hydrolases based on amino acid sequence similarities . The Biochemical Journal . 280 (Pt 2) . 2 . 309–16 . December 1991 . 1747104 . 1130547 . 10.1042/bj2800309 .
13. Islam MR, Tomatsu S, Shah GN, Grubb JH, Jain S, Sly WS . Active site residues of human β-glucuronidase. Evidence for Glu(540) as the nucleophile and Glu(451) as the acid-base residue . The Journal of Biological Chemistry . 274 . 33 . 23451–5 . August 1999 . 10438523 . 10.1074/jbc.274.33.23451 . free .
14. Wong AW, He S, Grubb JH, Sly WS, Withers SG . Identification of Glu-540 as the catalytic nucleophile of human beta-glucuronidase using electrospray mass spectrometry . The Journal of Biological Chemistry . 273 . 51 . 34057–62 . December 1998 . 9852062 . 10.1074/jbc.273.51.34057 . free .
15. Birkenmeier EH, Davisson MT, Beamer WG, Ganschow RE, Vogler CA, Gwynn B, Lyford KA, Maltais LM, Wawrzyniak CJ . Murine mucopolysaccharidosis type VII. Characterization of a mouse with beta-glucuronidase deficiency . The Journal of Clinical Investigation . 83 . 4 . 1258–66 . April 1989 . 2495302 . 303816 . 10.1172/JCI114010 . Muriel Davisson .
16. Haskins ME, Desnick RJ, DiFerrante N, Jezyk PF, Patterson DF . Beta-glucuronidase deficiency in a dog: a model of human mucopolysaccharidosis VII . Pediatric Research . 18 . 10 . 980–4 . October 1984 . 6436780 . 10.1203/00006450-198410000-00014 . free .
17. Fyfe JC, Kurzhals RL, Lassaline ME, Henthorn PS, Alur PR, Wang P, Wolfe JH, Giger U, Haskins ME, Patterson DF, Sun H, Jain S, Yuhki N . Molecular basis of feline beta-glucuronidase deficiency: an animal model of mucopolysaccharidosis VII . Genomics . 58 . 2 . 121–8 . June 1999 . 10366443 . 10.1006/geno.1999.5825 .
18. Marathe SV, McEwen JE . Vectors with the gus reporter gene for identifying and quantitating promoter regions in Saccharomyces cerevisiae . Gene . 154 . 1 . 105–7 . February 1995 . 7867935 . 10.1016/0378-1119(94)00845-J .
19. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE . UCSF Chimera--a visualization system for exploratory research and analysis . Journal of Computational Chemistry . 25 . 13 . 1605–12 . October 2004 . 15264254 . 10.1002/jcc.20084 . 8747218 .