Co-factor binding confers substrate specificity to xylose reductase from Debaryomyces hansenii.
Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards "non-substrate" sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (K(d)∼5.0-10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4-5 fold molar excess. Comparison of K(d) values with K(m) values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism.
Council of Scientific and Industrial Research (CSIR), Institute of Microbial Technology, Chandigarh, India.
Dipanwita
D
Biswas
Vaibhav
V
Pandya
Appu Kumar
AK
Singh
Alok K
AK
Mondal
S
S
Kumaran
D000449:Aldehyde Reductase / Q000737:chemistry / Q000378:metabolism*; D001051:Apoenzymes; D055162:Biocatalysis; D020134:Catalytic Domain; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009249:NADP / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012210:Rhamnose / Q000737:chemistry / Q000378:metabolism; D004718:Saccharomycetales / Q000737:chemistry / Q000201:enzymology*; D013379:Substrate Specificity; D014994:Xylose / Q000737:chemistry / Q000378:metabolism*
D001051:Apoenzymes; D003067:Coenzymes; D005656:Fungal Proteins; D020035:Holoenzymes; D011994:Recombinant Proteins; D009249:NADP; D014994:Xylose; D000449:Aldehyde Reductase; D012210:Rhamnose