Cupins are ubiquitous proteins existing in all three kingdoms of life and share a conserved b-barrel fold with a characteristic pocket located at the center of the b-barrel.1,2 Despite sharing the highly conserved topology, cupins show remarkable variations in their sequences, architecture of domains (comprising either one or two cupin domains), quaternary assembly, and the nature of bound metal ion as well.3 In the Pfam database (http://pfam.sanger.ac.uk/), cupins are classified into 35 protein families, with greatly diversified functions such as isomerases, epimerase, dioxygenase, nonenzymatic storage proteins, and it is suggested that cupin is one of the most functionally diverse protein superfamily.2,4 In recent years, large-scale sequencing of genomes and cDNAs has elicited a great number of hypothetical cupins whose biological functions need to be established. The cephalochordate amphioxus is a modern survivor of an ancient chordate lineage and acts as a living fossil between invertebrates and vertebrates. With the completion of the draft genome of Florida amphioxus Branchiostoma floridae in 2008, it was revealed that they are not only the most primitive chordates but also the genomic features appear to have a great deal in common with vertebrates as well.5 BbDUF985 is a hypothetical protein of 172 residues from Branchiostoma belcheri tsingtauense belonging to the cupin_5 family (Pfam entry: PF06172) of the cupin superfamily.4 Interestingly, although BbDUF985 is a protein from eukaryote, sequence homology analysis indicates that all its closest homologs are prokaryotic and more distantly kindred proteins from higher species. Previously, we solved the structure of YML079w (sharing 33% sequence identity with BbDUF985) from Saccharomyces cerevisiae in the guanosine triphosphate (GTP)-bound form,6 whereas the apo form structure is absent and further biochemical study was not undertook then. Here, we present the crystal structures of BbDUF985 in both the apo and guanosine diphosphate (GDP)-bound forms, which are the first pair of structures (with and without the ligand) from the cupin_5 family. Moreover, using fluorescence spectrometry, we determined the dissociation constant (KD) of GDP and investigated the spectra of BbDUF985 with several proposed ligands binding to the proteins in cupin superfamily. Combined with sequence analysis, we speculate that BbDUF985 and its homologs in the cupin_5 family might be involved in the nucleotide transport or metabolism.