Crystal structure of UDP‐galactose 4‐epimerase‐like l‐threonine dehydrogenase belonging to the intermediate short‐chain dehydrogenase‐reductase superfamily

Abstract

The crystal structure of a l‐threonine dehydrogenase (l‐ThrDH; EC 1.1.1.103) from the psychrophilic bacterium Flavobacterium frigidimaris KUC‐1, which shows no sequence similarity to conventional l‐ThrDHs, was determined in the presence of NAD and a substrate analog, glycerol. The asymmetric unit consisted of two subunits related by a two‐fold rotation axis. Each monomer consisted of a Rossmann‐fold domain and a carboxyl‐terminal catalytic domain. The overall fold of F. frigidimarisl‐ThrDH showed significant similarity to that of UDP‐galactose 4‐epimerase (GalE); however, structural comparison of the enzyme with E. coli and human GalEs showed clear topological differences in three loops (loop 1, loop 2 and the NAD‐binding loop) around the substrate and NAD binding sites. In F. frigidimarisl‐ThrDH, loops 1 and 2 insert toward the active site cavity, creating a barrier preventing the binding of UDP‐glucose. Alternatively, loop 1 contributes to a unique substrate binding pocket in the F. frigidimaris enzyme. The NAD binding loop, which tightly holds the adenine ribose moiety of NAD in the Escherichia coli and human GalEs, is absent in F. frigidimarisl‐ThrDH. Consequently, the cofactor binds to F. frigidimarisl‐ThrDH in a reversible manner, unlike its binding to GalE. The substrate binding model suggests that the reaction proceeds through abstraction of the β‐hydroxyl hydrogen of l‐threonine via either a proton shuttle mechanism driven by Tyr143 and facilitated by Ser118 or direct proton transfer driven by Tyr143. The present structure provides a clear bench mark for distinguishing GalE‐like l‐ThrDHs from GalEs. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8043828: l‐ThrDH (uniprotkb:http://www.uniprot.org/uniprot/Q8KZM4) and l‐ThrDH (uniprotkb:http://www.uniprot.org/uniprot/Q8KZM4) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by x‐ray crystallography (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114)

Binding of NAD+ and l-Threonine Induces Stepwise Structural and Flexibility Changes in Cupriavidus necator l-Threonine Dehydrogenase

“…The contacts between chains A and C and chains B and D represent native dimer contacts in SDR-like L-ThrDH. These contacts are also present in the previously determined structures of two other SDR-like L-ThrDHs, FfThrDH, and TvThrDH (16,17). Electron density was observed for amino acid residues 6 -313 in chains A, B, and C, and all of these residues could be assigned to the electron density map (Fig.…”

“…2-amino-3-keto butyrate). No previous report has described such a conduit within an SDR-like L-ThrDH in complex with NAD ϩ ; to date, all reported structures (16,17) are of closed forms, in which no such conduit is present.…”

“…SDR-like L-ThrDH can be divided into the NAD ϩ -binding and catalytic domains (16,17). In the CnThrDH structure, the NAD ϩ -binding domain consists of residues 1-175, 215-244, and 280 -300 (Fig.…”

The oxidative pentose phosphate pathway in the haloarchaeon Haloferax volcanii involves a novel type of glucose‐6‐phosphate dehydrogenase – The archaeal Zwischenferment

“…Based on conserved sequence motifs the majority of SDR proteins can be classified as “classical” and “extended” type [19] and at least four minor types can be defined [20]. An amino acid sequence alignment of H. volcanii Glc6PDH with the characterized SDRe members l ‐arabinose dehydrogenase ( l ‐AraDH) from H. volcanii [21], l ‐threonine dehydrogenase ( l ‐ThrDH) from Flavobacterium frigidimaris [22], dTDP‐glucose‐4,6‐dehydratase (GlcD) from H. volcanii [23] and UDP‐galactose‐4‐epimerase (GalE) from Pyrobaculum calidifontis [24] is shown in Fig. 4 .…”

Characterization of hypothetical protein VNG0128C fromHalobacteriumNRC-1 reveals GALE like activity and its involvement in Leloir pathway of galactose metabolism

“…Studies have also shown that L-threonine dehydrogenase from Flavobacterium frigidimaris shows significant sequence similarity with GALE while no sequence similarity with conventional L-threonine dehydrogenase (Yoneda, Sakuraba, Muraoka, Oikawa, & Ohshima, 2010). Hence, we performed a similar NADH monitoring assay in the presence of L-threonine, to verify if the protein can perform L-threonine dehydrogenase activity.…”