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 activesite Cys in plant THI4s and other indirect evidence implies that they are suicidal. To confirm this, we complemented the Arabidopsis tz-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 datamining 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. Short title: Suicidal and catalytic plant THI4 enzymes

The number of catalytic cycles in an enzyme’s lifetime and why it matters to metabolic engineering

“…Second, the overall lack of major differences between enzyme risk classes in terms of K d implies the steady-state level of failed enzymes would be more abundant for low CCR enzymes. Backing this interpretation, the suicide enzyme THI4 (CCR = 1) isolated from both yeast and Arabidopsis is mainly found in the failed state in vivo (17,67). Note that if b is generally the case, synthesis rates of damage-hardened enzymes might need to be tuned to take advantage of increased enzyme longevity.…”

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 (Δ thiG ) auxotrophic strain and prefers anaerobic, high-sulfide conditions, making it ill-suited for function in plant cells.…”

Potential For Applying Continuous Directed Evolution To Plant Enzymes

“…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,[33][34][35]. In contrast, certain prokaryotic THI4s are truly catalytic, i.e.…”

“…perform multiple reaction cycles; these enzymes use sulfide as sulfur source [36,37]. Thermovibrio ammonificans THI4 (TaTHI4) belongs to this group [35,38]. However, like other characterized prokaryotic THI4s, TaTHI4 prefers anaerobic, high-sulfide conditions, making it ill-suited for function in plant cells.…”