Hot or not? Discovery and characterization of a thermostable alditol oxidase from Acidothermus cellulolyticus 11B.
We describe the discovery, isolation and characterization of a highly thermostable alditol oxidase from Acidothermus cellulolyticus 11B. This protein was identified by searching the genomes of known thermophiles for enzymes homologous to Streptomyces coelicolor A3(2) alditol oxidase (AldO). A gene (sharing 48% protein sequence identity to AldO) was identified, cloned and expressed in Escherichia coli. Following 6xHis tag purification, characterization revealed the protein to be a covalent flavoprotein of 47 kDa with a remarkably similar reactivity and substrate specificity to that of AldO. A steady-state kinetic analysis with a number of different polyol substrates revealed lower catalytic rates but slightly altered substrate specificity when compared to AldO. Thermostability measurements revealed that the novel AldO is a highly thermostable enzyme with an unfolding temperature of 84 °C and an activity half-life at 75 °C of 112 min, prompting the name HotAldO. Inspired by earlier studies, we attempted a straightforward, exploratory approach to improve the thermostability of AldO by replacing residues with high B-factors with corresponding residues from HotAldO. None of these mutations resulted in a more thermostable oxidase; a fact that was corroborated by in silico analysis.
Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, Groningen, The Netherlands.
Remko T
RT
Winter
Dominic P H M
DP
Heuts
Egon M A
EM
Rijpkema
Edwin
E
van Bloois
Hein J
HJ
Wijma
Marco W
MW
Fraaije
D000192:Actinomycetales / Q000201:enzymology*; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D019295:Computational Biology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D048372:Streptomyces coelicolor / Q000235:genetics; D013379:Substrate Specificity; D013402:Sugar Alcohols / Q000378:metabolism*
D013402:Sugar Alcohols; D000429:Alcohol Oxidoreductases