The crystal structure of galactitol-1-phosphate 5-dehydrogenase from Escherichia coli K12 provides insights into its anomalous behavior on IMAC processes

• Published in: FEBS letters 2012-09, Vol.586 (19), p.3127-3133
• Main Authors: Esteban-Torres, María, Álvarez, Yanaisis, Acebrón, Iván, Rivas, Blanca de las, Muñoz, Rosario, Kohring, Gert-Wieland, Roa, Ana María, Sobrino, Mónica, Mancheño, José M.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Binding Sites; Chromatography, Affinity; Crystal structure; Crystallography, X-Ray; d-Tagatose-6-phosphate; Dehydrogenase; DNA, Bacterial - genetics; endogenous galactitol-1-phosphate 5-dehydrogenase; Enzyme Stability; Escherichia coli; Escherichia coli K12 - enzymology; Escherichia coli K12 - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Galactitol; Genes, Bacterial; GPDH; IMAC; immobilized metal affinity chromatography; Metal binding-site; Metals - metabolism; Models, Molecular; Molecular Sequence Data; nitrilotriacetic acid; NTA; Protein Multimerization; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; recombinant, untagged GPDH; rGPDH; Sequence Homology, Amino Acid; Sugar Alcohol Dehydrogenases - chemistry; Sugar Alcohol Dehydrogenases - genetics; Sugar Alcohol Dehydrogenases - metabolism; Zn-metalloenzyme
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2012.07.073

► rGPDH from E. coli spontaneously interact with NTA matrices. ► rGPDH is a homogeneous, stable dimer in solution. ► The presence of an N-terminal His6-tag in GPDH has adverse effects in its stability. ► rGPDHis an NAD(H)-dependent zinc metalloenzyme. ► The crystal structure of rGPDH has been solved up to 1.87Å resolution. Endogenous galactitol-1-phosphate 5-dehydrogenase (GPDH) (EC 1.1.1.251) from Escherichia coli spontaneously interacts with Ni2+-NTA matrices becoming a potential contaminant for recombinant, target His-tagged proteins. Purified recombinant, untagged GPDH (rGPDH) converted galactitol into tagatose, and d-tagatose-6-phosphate into galactitol-1-phosphate, in a Zn2+- and NAD(H)-dependent manner and readily crystallized what has permitted to solve its crystal structure. In contrast, N-terminally His-tagged GPDH was marginally stable and readily aggregated. The structure of rGPDH revealed metal-binding sites characteristic from the medium-chain dehydrogenase/reductase protein superfamily which may explain its ability to interact with immobilized metals. The structure also provides clues on the harmful effects of the N-terminal His-tag. GPDH and GPDHbind by molecular sieving (View interaction) GPDH and GPDHbind by x-ray crystallography(View interaction) GPDH and GPDHbind by cosedimentation in solution (View interaction)