Bioinformatic, genetic, and biochemical evidence that some glycoside hydrolase family 42 beta-galactosidases are arabinogalactan type I oligomer hydrolases.
Glycoside hydrolases are organized into glycoside hydrolase families (GHFs) and within this larger group, the beta-galactosidases are members of four families: 1, 2, 35, and 42. Most genes encoding GHF 42 enzymes are from prokaryotes unlikely to encounter lactose, suggesting a different substrate for these enzymes. In search of this substrate, we analyzed genes neighboring GHF 42 genes in databases and detected an arrangement implying that these enzymes might hydrolyze oligosaccharides released by GHF 53 enzymes from arabinogalactan type I, a pectic plant polysaccharide. Because Bacillus subtilis has adjacent GHF 42 and GHF 53 genes, we used it to test the hypothesis that a GHF 42 enzyme (LacA) could act on the oligosaccharides released by a GHF 53 enzyme (GalA) from galactan. We cloned these genes, plus a second GHF 42 gene from B. subtilis, yesZ, into Escherichia coli and demonstrated that cells expressing LacA with GalA gained the ability to use galactan as a carbon source. We constructed B. subtilis mutants and showed that the increased beta-galactosidase activity generated in response to the addition of galactan was eliminated by inactivating lacA or galA but unaffected by the inactivation of yesZ. As further demonstration, we overexpressed the LacA and GalA proteins in E. coli and demonstrated that these enzymes degrade galactan in vitro as assayed by thin-layer chromatography. Our work provides the first in vivo evidence for a function of some GHF 42 beta-galactosidases. Similar functions for other beta-galactosidases in both GHFs 2 and 42 are suggested by genomic data.
Shipkowski
Stephanie
S
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 209 South Frear, University Park, PA 16802, USA. sar242@psualum.com
Brenchley
Jean E
JE
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019295:Computational Biology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005685:Galactans / Q000145:classification / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000145:classification / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D005685:Galactans; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D001616:beta-Galactosidase; C005653:arabinogalactan