title: Isolation and divalent-metal activation of a β-xylosidase, RUM630-BX.
abstract: The gene encoding RUM630-BX, a β-xylosidase/arabinofuranosidase, was identified from activity-based screening of a cow rumen metagenomic library. The recombinant enzyme is activated as much as 14-fold (kcat) by divalent metals Mg(2+), Mn(2+) and Co(2+) but not by Ca(2+), Ni(2+), and Zn(2+). Activation of RUM630-BX by Mg(2+) (t0.5 144 s) is slowed two-fold by prior incubation with substrate, consistent with the X-ray structure of closely related xylosidase RS223-BX that shows the divalent-metal activator is at the back of the active-site pocket so that bound substrate could block its entrance. The enzyme is considerably more active on natural substrates than artificial substrates, with activity (kcat/Km) of 299 s(-1) mM(-1) on xylotetraose being the highest reported.
authors.affiliation: USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL 61604 USA.
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Jordan
authors.affiliation: USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL 61604 USA.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Braker
authors.affiliation: USDA-ARS-Western Regional Research Center, Albany, CA 94710 USA.
authors.forename: Kurt
authors.initials: K
authors.lastname: Wagschal
authors.affiliation: USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL 61604 USA.
authors.forename: J Rose
authors.initials: JR
authors.lastname: Stoller
authors.affiliation: USDA-ARS-Western Regional Research Center, Albany, CA 94710 USA. Electronic address: Charles.Lee@ars.usda.gov.
authors.forename: Charles C
authors.initials: CC
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle / Q000382:microbiology; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli; D006027:Glycosides / Q000378:metabolism; D056186:Metagenomics; D008969:Molecular Sequence Data; D009578:Nitrobenzenes / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012417:Rumen / Q000201:enzymology / Q000382:microbiology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014995:Xylosidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D006027:Glycosides; D009578:Nitrobenzenes; D011993:Recombinant Fusion Proteins; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase
keywords: Biofuels; Divalent metal activators; GH43 β-xylosidase; Highest k(cat)/K(m) (xylotetraose); Xylan