title: Engineering acidic Streptomyces rubiginosus D-xylose isomerase by rational enzyme design.
abstract: To maximize bioethanol production from lignocellulosic biomass, all sugars must be utilized. Yeast fermentation can be improved by introducing the d-xylose isomerase enzyme to convert the pentose sugar d-xylose, which cannot be fermented by Saccharomyces cerevisiae, into the fermentable ketose d-xylulose. The low activity of d-xylose isomerase, especially at the low pH required for optimal fermentation, limits its use. A rational enzyme engineering approach was undertaken, and seven amino acid positions were replaced to improve the activity of Streptomyces rubiginosus d-xylose isomerase towards its physiological substrate at pH values below 6. The active-site design was guided by mechanistic insights and the knowledge of amino acid protonation states at low pH obtained from previous joint X-ray/neutron crystallographic experiments. Tagging the enzyme with 6 or 12 histidine residues at the N-terminus resulted in a significant increase in the active-site affinity towards substrate at pH 5.8. Substituting an asparagine at position 215, which hydrogen bonded to the metal-bound Glu181 and Asp245, with an aspartate gave a variant with almost an order of magnitude lower KM than measured for the native enzyme, with a 4-fold increase in activity. Other studied variants showed similar (Asp57Asn, Glu186Gln/Asn215Asp), lower (Asp57His, Asn247Asp, Lys289His, Lys289Glu) or no (Gln256Asp, Asp287Asn, ΔAsp287) activity in acidic conditions relative to the native enzyme.
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
authors.forename: Mary Jo
authors.initials: MJ
authors.lastname: Waltman
authors.forename: Zamin Koo
authors.initials: ZK
authors.lastname: Yang
authors.forename: Paul
authors.initials: P
authors.lastname: Langan
authors.forename: David E
authors.initials: DE
authors.lastname: Graham
authors.forename: Andrey
authors.initials: A
authors.lastname: Kovalevsky
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020134:Catalytic Domain; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D011487:Protein Conformation; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D014994:Xylose / Q000378:metabolism
chemical_list: D014994:Xylose; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase
keywords: d-xylose isomerase; enzyme kinetics; protein engineering; rational design; site-directed mutagenesis