Immobilization of fumarase from thermophilic eukaryotic red alga Cyanidioschyzon merolae on ceramic carrier.

Fumarase is an enzyme catalyzing reversible reaction between fumarate and L-malate in the citric acid cycle. Fumarase is used in the industrial production of L-malate, and its immobilization is required for reuse of the fumarases to reduce the cost. Accordingly, understanding the properties of immobilized fumarase is crucial, and several groups report on the storage stability and kinetic parameters of immobilized fumarase. Here we have immobilized fumarase from the thermophilic red alga Cyanidioschyzon merolae (CmFUM) on ceramic beads and investigated its biochemical and physical properties. CmFUM demonstrated sufficient stability and reusability for industry use after immobilization. Notably, the thermostability was dramatically enhanced through immobilization. The Km value and kcat of immobilized CmFUM for fumarate were 1.7 mM and 22.7 s-1 respectively. The Km value for fumarate was lower than that of other reported immobilized fumarases, indicating a high substrate affinity of immobilized CmFUM. Furthermore, the enhanced stability resulting from immobilization partially compensated for the decrease in activity. The high affinity towards fumarate and good thermostability of immobilized CmFUM revealed in this study are advantageous traits for improving enzyme-mediated isomer-specific L-malate production.

Molecular and structural characterization of agmatine coumaroyltransferase in Triticeae, the key regulator of hydroxycinnamic acid amide accumulation.

Hydroxycinnamic acid amides (HCAAs) are involved in stress-induced defense in many plant species. Barley accumulates high concentrations of HCAAs irrespective of exogenous stressors, while other major cereals such as wheat and rice accumulate relatively low levels of HCAAs in intact tissues. The primary HCAA species in barley are biosynthesized by agmatine p-coumaroyltransferase (ACT), an N-acyltransferase of the BAHD superfamily. However, the molecular basis underlying barley's uniquely high HCAA accumulation has not been elucidated, and information regarding the structural details of BAHD N-acyltransferases is limited. Hence, we aimed to investigate the ACTs of family Poaceae. We isolated ACT (-like) genes, including those previously undescribed, and investigated their enzymatic and genetic features. All the identified enzymes belonged to clade IVa of the BAHD superfamily. The barley and wheat ACTs were further categorized, based on catalytic properties and primary structures, into ACT1 and ACT2 groups, the encoding loci of which are neighbors on the same chromosome. While all ACTs exhibited similar Km values for CoA-thioesters (acyl-group donors), members of the ACT1 group showed a distinctly higher affinity for agmatine (acyl-acceptor). Among the ACTs tested, an ACT isozyme in barley (HvACT1-1) showed the highest catalytic efficiency and transcript level, indicating that ACT regulates high-level HCAA accumulation in barley. For further enzymatic characterization of the ACTs, we crystalized wheat ACT2 (TaACT2) and determined its structure at 2.3 Å resolution. Structural alignment of TaACT2 and HvACT1-1 showed that the architectures of the substrate binding pockets were well conserved. However, the structure of a loop located at the entrance to acyl-acceptor binding site may be more flexible in TaACT2, which could be responsible for the lower affinity of TaACT2 to agmatine. Mutations of HvACT1-1 at Glu372 and Asp374 within one of the clade-IV specific motifs facing the deduced acyl-acceptor binding pocket caused significant catalytic deterioration toward agmatine both in Km and kcat, suggesting their key roles in acyl acceptor binding by the clade-IV enzymes. This study elucidated the molecular basis of how plants accumulate defensive specialized metabolites and provided insights into developing efficient and eco-friendly agricultural methods.

Crystal structure of barley agmatine coumaroyltransferase, an N-acyltransferase from the BAHD superfamily.

The enzymes of the BAHD superfamily, a large group of acyl-CoA-dependent acyltransferases in plants, are involved in the biosynthesis of diverse secondary metabolites. While the structures of several O-acyltransferases from the BAHD superfamily, such as hydroxycinnamoyl-CoA shikimate hydroxycinnamoyl transferase, have been elucidated, no structural information on N-acyltransferases is available. Hordeum vulgare agmatine coumaroyltransferase (HvACT) is an N-acyltransferase from the BAHD superfamily and is one of the most important enzymes in the secondary metabolism of barley. Here, an apo-form structure of HvACT is reported as the first structure of an N-acyltransferase from the BAHD superfamily. HvACT crystals diffracted to 1.8 Šresolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 57.6, b = 59.5, c = 73.6 Å, α = 90, ß = 91.3 , γ = 90°. Like other known BAHD superfamily structures, HvACT contains two domains that adopt a two-layer αß-sandwich architecture and a solvent-exposed channel that penetrates the enzyme core.