lacS (SSO3019) encodes the thermostable beta-glycosidase of the hyperthermophilic crenarchaeon Saccharolobus (Sulfolobus) solfataricus, a glycoside hydrolase family 1 (GH1, clan GH-A) enzyme with a (beta/alpha)8-barrel fold that assembles into a homotetramer. It is a retaining exo-glycosidase that uses Glu387 as the catalytic nucleophile and Glu206 as the general acid/base, and it removes terminal non-reducing beta-linked sugars with broad specificity: beta-D-glucosides and beta-D-fucosides are the preferred substrates, beta-D-galactosides (including lactose and X-Gal) are also hydrolysed, and beta-linked glucose di- and oligosaccharides such as cellobiose and cello-oligosaccharides are cleaved from the non-reducing end. In the cell the enzyme is intracellular and is required, together with the adjacent putative major-facilitator transporter LacTr, for growth on lactose; its transcription is controlled by a catabolite-repression-like system shared with other glycoside hydrolases. The enzyme is extremely thermostable, and in vivo lysine methylation contributes to that stability. It is widely used in biotechnology for glycoside hydrolysis and transglycosylation, for example to make galacto-oligosaccharides, and as a genetic reporter and selectable marker in Sulfolobus.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004553 hydrolase activity, hydrolyzing O-glycosyl compounds | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the GH1 signature IPR001360. LacS is a well-characterised GH1 retaining beta-glycosidase, so the parent term is correct but uninformative. Experimental work defines the specific activities: beta-glucosidase and beta-D-fucosidase (the preferred substrates) and beta-galactosidase, all at a common active site. Reason: The term is true but too general for an enzyme with experimentally defined substrate specificity. Grogan (1991) and Nucci et al. (1993) showed that the single enzyme hydrolyses beta-D-glucosides, beta-D-fucosides and beta-D-galactosides at one active site, with glucosides and fucosides preferred. The proposed replacements are is_a descendants of GO:0004553. Beta-galactosidase activity is already carried by the gene (GO:0004565). Proposed replacements: beta-glucosidase activity beta-galactosidase activity beta-D-fucosidase activity Supporting Evidence: PMID:16348503 Kinetic analyses indicated that chromogenic beta-d-galactosides and beta-d-glucosides are hydrolyzed at a common active site and that beta-glucosides and beta-fucosides represent the preferred substrates. PMID:8484908 has broad specificity for beta-D-gluco-, fuco- and galacto-sides PMID:8484908 From these results the enzyme from S. solfataricus strain MT-4 is defined as a true glycosyl hydrolase with remarkable exo-glucosidase activity |
| GO:0004565 beta-galactosidase activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC-mapping annotation from EC 3.2.1.23. The enzyme was first purified and cloned as the S. solfataricus beta-galactosidase. Its beta-galactosidase activity (o-nitrophenyl beta-galactoside, X-Gal, lactose) is established experimentally, and the Glu206 and Glu387 active-site mutants affect activity on aryl-galactosides. Reason: Beta-galactosidase activity is directly demonstrated for the purified native enzyme and underlies the lacS X-Gal colony screen. It is one of the enzyme's core hydrolytic activities, although beta-glucosides and beta-fucosides are kinetically preferred. Supporting Evidence: PMID:2105216 A thermophilic and thermostable beta-galactosidase activity was purified to homogeneity from crude extracts of the archaebacterium Sulfolobus solfataricus PMID:9010932 The Glu206-->Gln mutant showed 10- and 60-fold reduced activities on aryl-galacto and aryl-glucosides, respectively, when compared with the wild type. PMID:12813089 the S. solfataricus lacS gene ( 12 ) encodes a Ξ²-glycosidase (SsΞ²-gly) with broad substrate specificity, including activity against Ξ²-galactosides ( 26 ) and their chemical analogs, such as the chromogenic indicator X-Gal. |
| GO:0005975 carbohydrate metabolic process | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO process mapping from the GH1 signature. It is correct but very broad. Genetic evidence places LacS in lactose utilisation. A lacS-deficient deletion strain cannot grow on lactose, and restoring growth needs lacS together with the adjacent putative transporter gene lacTr. Reason: LacS catalyses the hydrolytic step of lactose breakdown itself (it cleaves the beta-galactosidic bond), so it passes the participation test for lactose catabolic process. This is the hydrolase role that also carries the term on the mammalian lactase PAINT node (PTN002570652, GO:0005990 IBD). Complementation of the lactose-negative deletion mutant needed both lacS and lacTr, which is consistent with uptake by LacTr followed by intracellular hydrolysis by LacS. The enzyme also hydrolyses cellobiose and cello-oligosaccharides in vitro, but no in vivo evidence assigns those substrates to it, so the replacement is limited to lactose catabolism. Proposed replacements: lactose catabolic process Supporting Evidence: PMID:12813089 The presence of both lacS and lacTr was necessary to sustain growth on the specific sugar and hence to complement the mutation PMID:12813089 the mutation remained totally nonpermissive for growth on lactose minimal medium PMID:10383958 acts at the level of transcription to coordinate synthesis of three physically unlinked glycosyl hydrolases implicated in carbohydrate utilization |
| GO:0008378 galactosyltransferase activity | IBA GO_REF:0000033 | REMOVE | Summary: IBA from PAINT node PTN008498319 in PTHR10353 (GH1). The IBD at this node is seeded only by Arabidopsis SFR2 (AT3G06510), a chloroplast outer-envelope galactolipid:galactolipid galactosyltransferase. SFR2 has lost detectable hydrolase activity. The same node also carries a negated IRD for GO:0008422 beta-glucosidase activity (PTHR10353-paint.tsv). PANTHER HMM scoring puts lacS and other archaeal GH1 beta-galactosidases (P14288, P50388, O52629) in subfamily SF209, which is named after SFR2. In QuickGO, lacS is the only non-plant protein among the 58 GO:0008378 IBA rows that cite this node. Reason: The galactosyltransferase activity placed at this node is a plant-specific neofunctionalisation. SFR2 evolved transferase activity by gaining a loop insertion, a C-terminal peptide and a hydrophobic patch, and it lost hydrolysis. The PAINT curator recorded that loss at the same node as a negated beta-glucosidase IRD. The node placement therefore claims a gain of transferase activity and a loss of hydrolase activity, and lacS contradicts both. It is experimentally a retaining hydrolase with strong beta-glucosidase, beta-fucosidase and beta-galactosidase activity, and it has no known role in galactolipid remodelling, which is a chloroplast-envelope process with no counterpart in archaeal ether-lipid membranes. Its close archaeal homologs bgaS and bglY in the same PANTHER subfamily receive beta-glucosidase, not galactosyltransferase, by TreeGrafter. That pattern suggests lacS reaches this node through subfamily assignment rather than by sharing the SFR2 innovation. In vitro, LacS (like all retaining GH1 enzymes) can transglycosylate and is used industrially to make galacto-oligosaccharides from lactose. That is a kinetic side reaction of the hydrolase mechanism under high substrate concentration, not the physiological galactosyl-transfer function the IBD asserts, so it does not rescue the annotation. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE WRONG ORTHOLOG OR PARALOG Sources checked: PANTHER:PTN008498319 Β· PTN008498319 SUPPORTS SOURCE BUT NOT TARGET The IBD is seeded only by SFR2 (AT3G06510), whose galactolipid galactosyltransferase activity with loss of hydrolysis is a plant neofunctionalisation. The node also carries a negated beta-glucosidase IRD. Of the 58 GO:0008378 IBA recipients, lacS is the only non-plant one, and it is an experimentally proven beta-glucosidase/beta-galactosidase hydrolase. Supporting Evidence: PMID:25100720 SFR2 has no detectable hydrolase activity, although its catalytic site is highly conserved with that of family 1 glycosyl hydrolases. PMID:20798281 SFR2 processively transfers galactosyl residues from the abundant monogalactolipid to different galactolipid acceptors PMID:16348503 beta-glucosides and beta-fucosides represent the preferred substrates PMID:9860840 hydrolyzes beta-glycosides following a retaining mechanism based upon the action of two amino acids |
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Download this section (compressed HTML)Q: Should PAINT node PTN008498319 (PTHR10353), which carries the SFR2-seeded galactosyltransferase IBD and a negated beta-glucosidase IRD, be restricted so that archaeal GH1 beta-galactosidases (lacS P22498, bgaS P14288, bglY P50388, O52629), which PANTHER HMMs place in subfamily SF209, are excluded?
Q: Which physiological beta-glycoside substrates (lactose, cello-oligosaccharides or other glycosides) does LacS hydrolyse in vivo, given that its kinetic preference in vitro is for beta-glucosides and beta-fucosides?
Experiment: Construct a clean lacS deletion in an otherwise wild-type background, leaving lacTr and xylS intact, and test growth on lactose, cellobiose and other beta-linked oligosaccharides, to define the in vivo substrate range of LacS separately from the adjacent transporter.
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