Molecular insight into the potential functional role of pseudoenzyme GFOD1 via interaction with NKIRAS2

Abstract

The glucose-fructose oxidoreductase/inositol dehydrogenase/rhizopine catabolism protein (Gfo/Idh/MocA) family includes a variety of oxidoreductases with a wide range of substrates that utilize NAD or NADP as redox cofactor. Human contains two members of this family, namely glucose-fructose oxidoreductase domain-containing protein 1 and 2 (GFOD1 and GFOD2). While GFOD1 exhibits low tissue specificity, it is notably expressed in the brain, potentially linked to psychiatric disorders and severe diseases. Nevertheless, the specific function, cofactor preference, and enzymatic activity of GFOD1 remain largely unknown. In this work, we find that GFOD1 does not bind to either NAD or NADP. Crystal structure analysis unveils that GFOD1 exists as a typical homodimer resembling other family members, but lacks essential residues required for cofactor binding, suggesting that it may function as a pseudoenzyme. Exploration of GFOD1-interacting partners in proteomic database identifies NF-κB inhibitor-interacting Ras-like 2 (NKIRAS2) as one potential candidate. Co-immunoprecipitation (co-IP) analysis indicates that GFOD1 interacts with both GTP- and GDP-bound forms of NKIRAS2. The predicted structural model of the GFOD1-NKIRAS2 complex is validated in cells using point mutants and shows that GFOD1 selectively recognizes the interswitch region of NKIRAS2. These findings reveal the distinct structural properties of GFOD1 and shed light on its potential functional role in cellular processes.

1. Introduction

Human glucose-fructose oxidoreductase domain-containing 1 (GFOD1), also known as C6orf114 or ADG-90, is a member of human GFOD family, along with another member, GFOD2. Notably, GFOD1 displays significant sequence homology with the bacterial enzyme glucose-fructose oxidoreductase (GFOR) [1]. GFOR belongs to the GFOR, inositol 2-dehydrogenase (IDH), and rhizopine catabolism protein MOCA (Gfo/Idh/MocA) family, which utilize NAD or NADP as a cofactor to catalyze dehydrogenation reaction of diverse substrates. The known structures of members of this family are predominantly found in dimeric or tetrameric form. The tetrameric quaternary structure of Zymomonas mobilis GFOR (ZmGFOR) reveals that each subunit comprises two domains: an N-terminal classical dinucleotide-binding domain bearing a Rossmann α/β-fold, and a C-terminal domain consisting of a large “open-faced” β-sheet of eight mostly antiparallel β-strands [2–4]. Functionally, GFOR catalyzes the oxidation of glucose to gluconolactone which is rapidly hydrolyzed to gluconic acid, and the reduction of fructose to sorbitol, in order to overcome osmotic stress in high external sugar concentrations [5–7]. Although GFOD1 is homologous to GFOR, the potential catalytic activity of GFOD1 for analogous reactions remains to be investigated.

So far, limited information about the function of GFOD1 is available. RNA-seq investigation indicated that GFOD1 exhibits low tissue specificity in a majority of human tissues, with the brain showing the highest expression, followed by the esophagus, bone marrow, heart, spleen, and adipose tissues [8]. In zebrafish, both GFOD1 and GFOD2 display similar expression patterns in the developing and mature central nervous systems, with GFOD1 exhibiting higher expression than GFOD2 [1]. Along with its high expression in the brain, GFOD1 is closely linked to psychiatric disorders such as attention-deficit/hyperactivity disorder (ADHD), autism spectrum disorder (ASD) and schizophrenia. Elevated GFOD1 expression was observed in mice with genetic defects associated with ADHD and ASD, as well as in the hippocampal tissue of patients with ADHD and temporal lobe epilepsy (TLE) [9–11]. In addition, a single nucleotide polymorphism (SNP) rs552655 in GFOD1 is associated with inattention symptoms in ADHD, as identified through whole-genome association studies [10,12,13]. Moreover, a meta-analysis linked GFOD1 to a linkage region associated with schizophrenia, suggesting a possible contribution to the risk of schizophrenia in diverse populations [14].

Several studies have also investigated the association of GFOD1 with various severe conditions. Notably, GFOD1 mRNA was found to be significantly elevated in clear cell renal cell carcinoma (ccRCC), showing a positive correlation with histological grade and stage [15]. Additionally, a fusion of GFOD1 with neurotrophic tyros...