A single amino acid change (Y318F) in the L-arabitol dehydrogenase (LadA) from Aspergillus niger results in a significant increase in affinity for D-sorbitol.
BACKGROUND
L-arabitol dehydrogenase (LAD) and xylitol dehydrogenase (XDH) are involved in the degradation of L-arabinose and D-xylose, which are among the most abundant monosaccharides on earth. Previous data demonstrated that LAD and XDH not only differ in the activity on their biological substrate, but also that only XDH has significant activity on D-sorbitol and may therefore be more closely related to D-sorbitol dehydrogenases (SDH). In this study we aimed to identify residues involved in the difference in substrate specificity.


RESULTS
Phylogenetic analysis demonstrated that LAD, XDH and SDH form 3 distinct groups of the family of dehydrogenases containing an Alcohol dehydrogenase GroES-like domain (pfam08240) and likely have evolved from a common ancestor. Modelling of LadA and XdhA of the saprobic fungus Aspergillus niger on human SDH identified two residues in LadA (M70 and Y318), that may explain the absence of activity on D-sorbitol. While introduction of the mutation M70F in LadA of A. niger resulted in a nearly complete enzyme inactivation, the Y318F resulted in increased activity for L-arabitol and xylitol. Moreover, the affinity for D-sorbitol was increased in this mutant.


CONCLUSIONS
These data demonstrates that Y318 of LadA contributes significantly to the substrate specificity difference between LAD and XDH/SDH.
Rutten
Lucy
L
Department of Crystal and Structural Chemistry, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands. lucy.rutten@gmail.com
Ribot
Cecile
C
Trejo-Aguilar
Blanca
B
Wösten
Han A B
HA
de Vries
Ronald P
RP
D019943:Amino Acid Substitution; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D050542:D-Xylulose Reductase / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D007064:L-Iditol 2-Dehydrogenase / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D013012:Sorbitol / Q000378:metabolism*; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics / Q000378:metabolism*
D004271:DNA, Fungal; D013012:Sorbitol; D013401:Sugar Alcohol Dehydrogenases; D007064:L-Iditol 2-Dehydrogenase; D050542:D-Xylulose Reductase
Microbiology (Reading). 2003 May;149(Pt 5):1183-1191
12724380
Electrophoresis. 1997 Dec;18(15):2714-23
9504803
Microbiol Mol Biol Rev. 2001 Dec;65(4):497-522, table of contents
11729262
Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387-95
6546423
Chem Biol Interact. 2001 Jan 30;130-132(1-3):351-8
11306057
Eur J Biochem. 2004 May;271(10):1864-72
15128296
Nucleic Acids Res. 2003 Jul 1;31(13):3381-5
12824332
FEMS Microbiol Lett. 1994 Oct 15;123(1-2):83-90
7988903
Curr Protoc Bioinformatics. 2002 Aug;Chapter 2:Unit 2.3
18792934
J Biol Chem. 2001 Nov 2;276(44):40631-7
11514550
Nucleic Acids Res. 1988 Aug 11;16(15):7583-600
2970625
Eukaryot Cell. 2003 Oct;2(5):867-75
14555469
Biotechnol Prog. 2005 Nov-Dec;21(6):1610-6
16321042
J Mol Biol. 1990 Oct 5;215(3):403-10
2231712
Eur J Biochem. 2001 Oct;268(20):5414-23
11606204
Structure. 2003 Sep;11(9):1071-85
12962626