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

AbstractEndogenous 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.Structured summary of protein interactionsGPDH and GPDH bind by molecular sieving (View interaction)GPDH and GPDH bind by x-ray crystallography(View interaction)GPDH and GPDH bind by cosedimentation in solution (View interaction

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

Endogenous galactitol-1-phosphate 5-dehydrogenase (GPDH) (EC 1.1.1.251) from Escherichia coli spontaneously interacts with Ni 2+-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 Zn 2+- 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. Structured summary of protein interactions: GPDH and GPDH bind by molecular sieving (View interaction) GPDH and GPDH bind by x-ray crystallography (View interaction) GPDH and GPDH bind by cosedimentation in solution (View interaction). © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.JMM and RM thank the Ministerio de Ciencia e Innovación for their research grantsBFU2010-17929, CSD2006-00015 and AGL2011-22745, CSD2007-00063 FUN-C-FOOD and Comunidad de Madrid S2009/AGR-1469, respectively.Peer Reviewe