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β-Galactosidase

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β-Galactosidase
β-galactosidase fromPenicillium sp.
Identifiers
EC no.3.2.1.23
CAS no.9031-11-2
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDBstructuresRCSB PDBPDBePDBsum
Gene OntologyAmiGO/QuickGO
Search
PMCarticles
PubMedarticles
NCBIproteins
galactosidase, beta 1
Identifiers
SymbolGLB1
Alt. symbolsELNR1
NCBI gene2720
HGNC4298
OMIM230500
RefSeqNM_000404
UniProtP16278
Other data
LocusChr. 3p22.3
Search for
StructuresSwiss-model
DomainsInterPro

β-Galactosidase(EC 3.2.1.23,beta-galorβ-gal;systematic nameβ-D-galactoside galactohydrolase) is aglycoside hydrolaseenzymethatcatalyzeshydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. (This enzyme digests many β-Galactosides, not just lactose. It is sometimes loosely referred to aslactasebut that name is generally reserved for mammalian digestive enzymes that breaks down lactose specifically.)

β-Galactosides include carbohydrates containinggalactosewhere the glycosidic bond lies above the galactose molecule.Substratesof different β-galactosidases includegangliosideGM1, lactosylceramides,lactose,and variousglycoproteins.[1]

Function

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β-Galactosidase is anexoglycosidasewhich hydrolyzes the β-glycosidic bondformed between agalactoseand its organic moiety. It may also cleavefucosidesandarabinosidesbut at a much lower rate. It is an essential enzyme in the human body. Deficiencies in the protein can result ingalactosialidosisorMorquio B syndrome.InE. coli,thelacZgene is the structural gene for β-galactosidase; which is present as part of the inducible systemlacoperonwhich is activated in the presence oflactosewhenglucoselevel is low. β-Galactosidase synthesis stops when glucose levels are sufficient.[2]

β-Galactosidase has manyhomologuesbased on similar sequences. A few are evolved β-galactosidase (EBG),β-glucosidase,6-phospho-β-galactosidase, β-mannosidase, and lactase-phlorizin hydrolase. Although they may be structurally similar, they all have different functions.[3]Beta-gal is inhibited byL-riboseand bycompetitive inhibitors2-phenylethyl 1-thio-β-D-galactopyranoside (PETG),D-galactonolactone,isopropyl thio-β-D-galactoside (IPTG),and galactose.[4]

β-Galactosidase is important for organisms as it is a key provider in the production of energy and a source of carbons through the break down of lactose to galactose and glucose. It is also important forlactose-intolerantpeople as it is responsible for making lactose-free milk and other dairy products. Many adult humans lack thelactaseenzyme, which has the same function as β-galactosidase, so they are not able to properly digest dairy products. β-Galactose is used in such dairy products as yogurt, sour cream, and some cheeses which are treated with the enzyme to break down any lactose before human consumption. In recent years, β-galactosidase has been researched as a potential treatment for lactose intolerance through gene replacement therapy where it could be placed into the human DNA so individuals can break down lactose on their own.[5][6]

Structure

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The 1,023amino acidsofE. coliβ-galactosidase were sequenced in 1983,[7]and its structure determined eleven years later in 1994. Theproteinis a 464-kDahomotetramerwith 2,2,2-pointsymmetry.[8]Each unit of β-galactosidase consists of fivedomains;domain 1 is a jelly-roll typeβ-barrel,domain 2 and 4 arefibronectin type III-like barrels, domain 5 a novel β-sandwich, while the central domain 3 is a distortedTIM-type barrel,lacking the fifth helix with a distortion in the sixth strand.[8]

The third domain contains the active site.[9]The active site is made up of elements from two subunits of the tetramer, and disassociation of the tetramer into dimers removes critical elements of the active site. The amino-terminal sequence of β-galactosidase, the α-peptide involved in α-complementation, participates in a subunit interface. Its residues 22–31 help to stabilize a four-helix bundle which forms the major part of that interface, and residue 13 and 15 also contributing to the activating interface.[citation needed]These structural features provide a rationale for the phenomenon of α-complementation, where the deletion of the amino-terminal segment results in the formation of an inactive dimer.

Reaction

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β-galactosidase reaction

β-Galactosidase can catalyze three different reactions in organisms. In one, it can go through a process called transgalactosylation to makeallolactose,creating apositive feedback loopfor the production of β-galactose. Allolactose can also be cleaved to form monosaccharides. It can also hydrolyze lactose intogalactoseandglucosewhich will proceed intoglycolysis.[3]The active site of β-galactosidase catalyzes the hydrolysis of itsdisaccharidesubstrate via "shallow" (nonproductive site) and "deep" (productive site) binding.Galactosidessuch as PETG and IPTG will bind in the shallow site when the enzyme is in "open" conformation whiletransition state analogssuch asL-ribose andD-galactonolactone will bind in the deep site when the conformation is "closed".[4]

The enzymatic reaction consists of two chemical steps, galactosylation and degalactosylation. Galactosylation is the first chemical step in the reaction where Glu461 donates a proton to a glycosidic oxygen, resulting in galactose covalently bonding with Glu537. In the second step, degalactosylation, the covalent bond is broken when Glu461 accepts a proton, replacing the galactose with water. Twotransition statesoccur in the deep site of the enzyme during the reaction, once after each step. When water participates in the reaction, galactose is formed, otherwise, whenD-glucose acts as the acceptor in the second step, transgalactosylation occurs.[4]It has been kinetically measured that single tetramers of the protein catalyze reactions at a rate of 38,500 ± 900 reactions per minute.[10]Monovalentpotassiumions(K+) as well as divalentmagnesiumions (Mg2+) are required for the enzyme's optimal activity. The β-linkage of the substrate is cleaved by a terminalcarboxylgroup on theside chainof aglutamic acid.

The image on the left is aribbon diagramof beta-galactosidase displaying the location of Glu 461, Glu 537, and Gly 794. The image on the right is a zoomed in version showing the interaction between the amino acids.

InE. coli,Glu-461 was thought to be thenucleophilein thesubstitutionreaction.[11]However, it is now known that Glu-461 is anacidcatalyst. Instead, Glu-537 is the actual nucleophile,[12]binding to a galactosyl intermediate. Inhumans,thenucleophileof thehydrolysisreaction is Glu-268.[13]Gly794 is important for β-galactosidase activity. It is responsible for putting the enzyme in a "closed", ligand bounded, conformation or "open" conformation, acting like a "hinge" for the active site loop. The different conformations ensure that only preferential binding occurs in the active site. In the presence of a slow substrate, Gly794 activity increased as well as an increase in galactosylation and decrease in degalactosylation.[4]

Applications

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The β-galactosidase assay is used frequently ingenetics,molecular biology,and otherlife sciences.[14]An active enzyme may be detected using artificial chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside,X-gal.β-galactosidase will cleave the glycosidic bond inX-galand form galactose and 5-bromo-4-chloro-3-hydroxyindole which dimerizes and oxidizes to 5,5'-dibromo-4,4'-dichloro-indigo, an intense blue product that is easy to identify and quantify.[15]It is used for example inblue white screen.[16]Its production may be induced by a non-hydrolyzableanalogofallolactose,IPTG,which binds and releases the lac repressor from the lac operator, thereby allowing the initiation of transcription to proceed.

It is commonly used in molecular biology as areporter markerto monitor gene expression. It also exhibits a phenomenon called α-complementation which forms the basis for theblue/white screeningof recombinant clones. This enzyme can be split in two peptides, LacZαand LacZΩ,neither of which is active by itself but when both are present together, spontaneously reassemble into a functional enzyme. This property is exploited in manycloning vectorswhere the presence of thelacZαgene in a plasmid can complement intransanother mutant gene encoding the LacZΩ in specific laboratory strains ofE. coli.However, when DNA fragments are inserted in the vector, the production of LacZα is disrupted, the cells therefore show no β-galactosidase activity. The presence or absence of an active β-galactosidase may be detected byX-gal,which produces a characteristic blue dye when cleaved by β-galactosidase, thereby providing an easy means of distinguishing the presence or absence of cloned product in a plasmid. In studies of leukaemia chromosomal translocations, Dobson and colleagues used a fusion protein of LacZ in mice,[17]exploiting β-galactosidase's tendency to oligomerise to suggest a potential role for oligomericity in MLL fusion protein function.[18]

A recent study conducted in 2020–2021 determined that Beta-Galactosidase activity correlates with senescence of the cells. Senescence of the cells can be interpreted as cells that do not divide, but cells that do not die. Beta-Galactosidase activity can be overexpressed, and this can lead to various diseases afflicting a wide range of body systems. These systems include the cardiovascular system, skeletal system, and many more. Detecting senescence cells can be achieved by measuring the lysosomal Beta-Galactosidase activity.[19]

A new isoform for beta-galactosidase with optimum activity at pH 6.0 (Senescence Associated beta-gal orSA-beta-gal)[20]which is specifically expressed insenescence(the irreversible growth arrest of cells). Specific quantitative assays were even developed for its detection.[21][15][22]However, it is now known that this is due to an overexpression and accumulation of the lysosomal endogenous beta-galactosidase,[23]and its expression is not required for senescence. Nevertheless, it remains the most widely used biomarker for senescent and aging cells, because it is reliable and easy to detect.

Evolution

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Some species of bacteria, includingE. coli,have additional β-galactosidase genes. A second gene, called evolved β-galactosidase (ebgA) gene was discovered when strains with thelacZgene deleted (but still containing the gene for galactoside permease,lacY), were plated on medium containing lactose (or other 3-galactosides) as sole carbon source. After a time, certain colonies began to grow. However, the EbgA protein is an ineffective lactase and does not allow growth on lactose. Two classes of single point mutations dramatically improve the activity of ebg enzyme toward lactose.[24][25]and, as a result, the mutant enzyme is able to replace the lacZ β-galactosidase.[26]EbgA and LacZ are 50% identical on the DNA level and 33% identical on the amino acid level.[27]The active ebg enzyme is an aggregate of ebgA -gene and ebgC-gene products in a 1:1 ratio with the active form of ebg enzymes being anα4 β4 hetero-octamer.[28]

Species distribution

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Much of the work done on β-galactosidase is derived fromE. coli.However the enzyme can be found in many plants (especially fruits), mammals, yeast, bacteria, and fungi.[29]β-galactosidase genes can differ in the length of their coding sequence and the length of proteins formed by amino acids.[30]This separates the β-galactosidases into four families: GHF-1, GHF-2, GHF-35, and GHF- 42.[31]E. Colibelongs to GHF-2, all plants belong to GHF-35, andThermus thermophilusbelongs to GHF-42.[31][30]Various fruits can express multiple β-galactosidase genes. There are at least seven β-galactosidase genes expressed in tomato fruit development, that have amino acid similarity between 33% and 79%.[32]A study targeted at identifying fruit softening of peaches found 17 different gene expressions of β-galactosidases.[30]The only other known crystal structure of β-galactosidase is fromThermus thermophilus.[31]

References

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  4. ^abcdJuers DH, Hakda S, Matthews BW, Huber RE (November 2003). "Structural basis for the altered activity of Gly794 variants ofEscherichia coliβ-galactosidase ".Biochemistry.42(46): 13505–11.doi:10.1021/bi035506j.PMID14621996.
  5. ^Salehi S, Eckley L, Sawyer GJ, Zhang X, Dong X, Freund JN, Fabre JW (January 2009). "Intestinal lactase as an autologous β-galactosidase reporter gene for in vivo gene expression studies".Human Gene Therapy.20(1): 21–30.doi:10.1089/hum.2008.101.PMID20377368.
  6. ^Ishikawa K, Kataoka M, Yanamoto T, Nakabayashi M, Watanabe M, Ishihara S, Yamaguchi S (July 2015)."Crystal structure of β-galactosidase fromBacillus circulansATCC 31382 (BgaD) and the construction of the thermophilic mutants ".The FEBS Journal.282(13): 2540–52.doi:10.1111/febs.13298.PMID25879162.S2CID33928719.
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  11. ^Gebler JC, Aebersold R, Withers SG (June 1992)."Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) β-galactosidase fromEscherichia coli".The Journal of Biological Chemistry.267(16): 11126–30.doi:10.1016/S0021-9258(19)49884-0.PMID1350782.
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  13. ^McCarter JD, Burgoyne DL, Miao S, Zhang S, Callahan JW, Withers SG (January 1997)."Identification of Glu-268 as the catalytic nucleophile of human lysosomal β-galactosidase precursor by mass spectrometry"(PDF).The Journal of Biological Chemistry.272(1): 396–400.doi:10.1074/jbc.272.1.396.PMID8995274.S2CID35101194.
  14. ^Ninfa AJ, Ballou DP (2009).Fundamental Laboratory Approaches for Biochemistry and Biotechnology.ISBN978-0-470-47131-9.
  15. ^abGary RK, Kindell SM (August 2005). "Quantitative assay of senescence-associated beta-galactosidase activity in mammalian cell extracts".Analytical Biochemistry.343(2): 329–34.doi:10.1016/j.ab.2005.06.003.PMID16004951.
  16. ^β-Galactosidase Assay (A better Miller) - OpenWetWare
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  19. ^Lozano-Torres, Beatriz; Blandez, Juan F.; Sancenón, Félix; Martínez-Máñez, Ramón (April 2021)."Chromo-fluorogenic probes for β-galactosidase detection".Analytical and Bioanalytical Chemistry.413(9): 2361–2388.doi:10.1007/s00216-020-03111-8.hdl:10251/180327.ISSN1618-2642.PMID33606064.S2CID231957317.
  20. ^Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, et al. (September 1995)."A biomarker that identifies senescent human cells in culture and in aging skin in vivo".Proceedings of the National Academy of Sciences of the United States of America.92(20): 9363–7.Bibcode:1995PNAS...92.9363D.doi:10.1073/pnas.92.20.9363.PMC40985.PMID7568133.
  21. ^Bassaneze V, Miyakawa AA, Krieger JE (January 2008). "A quantitative chemiluminescent method for studying replicative and stress-induced premature senescence in cell cultures".Analytical Biochemistry.372(2): 198–203.doi:10.1016/j.ab.2007.08.016.PMID17920029.
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  23. ^Lee BY, Han JA, Im JS, Morrone A, Johung K, Goodwin EC, et al. (April 2006). "Senescence-associated β-galactosidase is lysosomal β-galactosidase".Aging Cell.5(2): 187–95.doi:10.1111/j.1474-9726.2006.00199.x.hdl:2158/216175.PMID16626397.S2CID82432911.
  24. ^Hall BG (January 1977)."Number of mutations required to evolve a new lactase function inEscherichia coli".Journal of Bacteriology.129(1): 540–3.doi:10.1128/JB.129.1.540-543.1977.PMC234956.PMID318653.
  25. ^Hall BG (July 1981). "Changes in the substrate specificities of an enzyme during directed evolution of new functions".Biochemistry.20(14): 4042–9.doi:10.1021/bi00517a015.PMID6793063.
  26. ^Hall BG (October 1976). "Experimental evolution of a new enzymatic function. Kinetic analysis of the ancestral (ebg) and evolved (ebg) enzymes".Journal of Molecular Biology.107(1): 71–84.doi:10.1016/s0022-2836(76)80018-6.PMID794482.
  27. ^Stokes HW, Betts PW, Hall BG (November 1985)."Sequence of the ebgA gene ofEscherichia coli:comparison with the lacZ gene ".Molecular Biology and Evolution.2(6): 469–77.doi:10.1093/oxfordjournals.molbev.a040372.PMID3939707.
  28. ^Elliott AC, K S, Sinnott ML, Smith PJ, Bommuswamy J, Guo Z, et al. (February 1992)."The catalytic consequences of experimental evolution. Studies on the subunit structure of the second (ebg) β-galactosidase ofEscherichia coli,and on catalysis by ebgab, an experimental evolvant containing two amino acid substitutions ".The Biochemical Journal.282 ( Pt 1) (1): 155–64.doi:10.1042/bj2820155.PMC1130902.PMID1540130.
  29. ^Richmond ML, Gray JI, Stine CM (1981)."β-Galactosidase: Review of Recent Research Related to Technological Application, Nutritional Concerns, and Immobilization".Journal of Dairy Science.64(9): 1759–1771.doi:10.3168/jds.s0022-0302(81)82764-6.ISSN0022-0302.
  30. ^abcGuo S, Song J, Zhang B, Jiang H, Ma R, Yu M (2018). "Genome-wide identification and expression analysis of β-galactosidase family members during fruit softening of peach [Prunus persica(L.) Batsch] ".Postharvest Biology and Technology.136:111–123.doi:10.1016/j.postharvbio.2017.10.005.
  31. ^abcRojas AL, Nagem RA, Neustroev KN, Arand M, Adamska M, Eneyskaya EV, et al. (November 2004). "Crystal structures of β-galactosidase fromPenicilliumsp. and its complex with galactose ".Journal of Molecular Biology.343(5): 1281–92.doi:10.1016/j.jmb.2004.09.012.PMID15491613.
  32. ^Smith DL, Gross KC (July 2000)."A family of at least seven β-galactosidase genes is expressed during tomato fruit development".Plant Physiology.123(3): 1173–83.doi:10.1104/pp.123.3.1173.PMC59080.PMID10889266.
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