Bioinformatic and experimental evidence for suicidal and catalytic plant THI4s

Abstract

Like fungi and some prokaryotes, plants use a thiazole synthase (THI4) to make the thiazole precursor of thiamin. Fungal THI4s are suicide enzymes that destroy an essential active-site Cys residue to obtain the sulfur atom needed for thiazole formation. In contrast, certain prokaryotic THI4s have no active-site Cys, use sulfide as sulfur donor, and are truly catalytic. The presence of a conserved active-site Cys in plant THI4s and other indirect evidence implies that they are suicidal. To confirm this, we complemented the Arabidopsistz-1 mutant, which lacks THI4 activity, with a His-tagged Arabidopsis THI4 construct. LC–MS analysis of tryptic peptides of the THI4 extracted from leaves showed that the active-site Cys was predominantly in desulfurated form, consistent with THI4 having a suicide mechanism in planta. Unexpectedly, transcriptome data mining and deep proteome profiling showed that barley, wheat, and oat have both a widely expressed canonical THI4 with an active-site Cys, and a THI4-like paralog (non-Cys THI4) that has no active-site Cys and is the major type of THI4 in developing grains. Transcriptomic evidence also indicated that barley, wheat, and oat grains synthesize thiamin de novo, implying that their non-Cys THI4s synthesize thiazole. Structure modeling supported this inference, as did demonstration that non-Cys THI4s have significant capacity to complement thiazole auxotrophy in Escherichia coli. There is thus a prima facie case that non-Cys cereal THI4s, like their prokaryotic counterparts, are catalytic thiazole synthases. Bioenergetic calculations show that, relative to suicide THI4s, such enzymes could save substantial energy during the grain-filling period.

Antioxidants in Potatoes: A Functional View on One of the Major Food Crops Worldwide

“…HET-P is formed from NAD and glycine by HET-P synthase (THI1), which provides the necessary sulfur from an active-site cysteine, and is thus a single-turnover reaction [ 39 ]. True catalytic THI1 paralogs that do not contain an active-site cysteine but use sulfide as sulfur donor have recently been identified in some cereals [ 39 ]. TMP is then dephosphorylated to thiamin by TMP phosphatase TH2 in the cytosol or mitochondria [ 40 , 41 ].…”

Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study

“…Plant and yeast THI4s are suicide enzymes that self-inactivate after a single catalytic cycle because they obtain the sulfur atom needed to form the thiazole product by destroying an active-site cysteine residue. Such THI4s are consequently energetically expensive to operate [3,[34][35][36]. In contrast, certain prokaryotic THI4s are truly catalytic, i.e., perform multiple reaction cycles; these enzymes use sulfide as sulfur source ( Figure S1) [37,38].…”

“…In contrast, certain prokaryotic THI4s are truly catalytic, i.e., perform multiple reaction cycles; these enzymes use sulfide as sulfur source ( Figure S1) [37,38]. Thermovibrio ammonificans THI4 (TaTHI4) belongs to this group [36,39]. However, similar to certain other prokaryotic THI4s previously characterized by our group [39], TaTHI4 has only low complementing activity in an E. coli thiazole (∆thiG) auxotrophic strain and prefers anaerobic, high-sulfide conditions, making it ill-suited for function in plant cells.…”

Structure and function of aerotolerant, multiple-turnover THI4 thiazole synthases

“…Plants, fungi, and some prokaryotes make ADT via the thiazole synthase THI4, a single-turnover suicide enzyme [ 3–6 ]. In a reaction requiring iron (yeast) or zinc ( Arabidopsis ), these THI4s form ADT from NAD, glycine, and a sulfur atom stripped from an active-site Cys residue [ 3 , 5 , 7 , 8 ]. The sulfur loss converts Cys to dehydroalanine and irreversibly inactivates the enzyme [ 3 , 5 ] ( Figure 1 ).…”

“…In a reaction requiring iron (yeast) or zinc ( Arabidopsis ), these THI4s form ADT from NAD, glycine, and a sulfur atom stripped from an active-site Cys residue [ 3 , 5 , 7 , 8 ]. The sulfur loss converts Cys to dehydroalanine and irreversibly inactivates the enzyme [ 3 , 5 ] ( Figure 1 ). Such THI4s must, therefore, be replaced after just one reaction cycle, and this — plus the high demand for thiazole [ 9 ] — makes THI4 one of the shortest-lived proteins in plant leaves [ 10 , 11 ].…”