Identification and Characterization of the Missing Pyrimidine Reductase in the Plant Riboflavin Biosynthesis Pathway    

Abstract

Riboflavin (vitamin B2) is the precursor of the flavin coenzymes flavin mononucleotide and flavin adenine dinucleotide. In Escherichia coli and other bacteria, sequential deamination and reduction steps in riboflavin biosynthesis are catalyzed by RibD, a bifunctional protein with distinct pyrimidine deaminase and reductase domains. Plants have two diverged RibD homologs, PyrD and PyrR; PyrR proteins have an extra carboxyl-terminal domain (COG3236) of unknown function. Arabidopsis (Arabidopsis thaliana) PyrD (encoded by At4g20960) is known to be a monofunctional pyrimidine deaminase, but no pyrimidine reductase has been identified. Bioinformatic analyses indicated that plant PyrR proteins have a catalytically competent reductase domain but lack essential zinc-binding residues in the deaminase domain, and that the Arabidopsis PyrR gene (At3g47390) is coexpressed with riboflavin synthesis genes. These observations imply that PyrR is a pyrimidine reductase without deaminase activity. Consistent with this inference, Arabidopsis or maize (Zea mays) PyrR (At3g47390 or GRMZM2G090068) restored riboflavin prototrophy to an E. coli ribD deletant strain when coexpressed with the corresponding PyrD protein (At4g20960 or GRMZM2G320099) but not when expressed alone; the COG3236 domain was unnecessary for complementing activity. Furthermore, recombinant maize PyrR mediated NAD(P)H-dependent pyrimidine reduction in vitro. Import assays with pea (Pisum sativum) chloroplasts showed that PyrR and PyrD are taken up and proteolytically processed. Ablation of the maize PyrR gene caused early seed lethality. These data argue that PyrR is the missing plant pyrimidine reductase, that it is plastid localized, and that it is essential. The role of the COG3236 domain remains mysterious; no evidence was obtained for the possibility that it catalyzes the dephosphorylation that follows pyrimidine reduction.

A directed-overflow and damage-control N-glycosidase in riboflavin biosynthesis

“…This strain is auxotrophic for riboflavin in absence of the inducer anhydrotetracycline. The E. coli ribA gene and the gene encoding the V. vulnificus COG3236-RibA fusion protein were PCR-amplified from pET30a-RibA [13] and genomic DNA, respectively. The forward primers contained a KpnI site and a Shine-Dalgarno sequence before the start codon; the reverse primers contained an SphI site after the stop codon.…”

“…The COG3236 domains of the Arabidopsis and maize RIBR genes were amplified from pGEM-4Z-RIBR constructs [13]. Amplicons were cloned into pET28b, adding an N-terminal His-tag, and the resulting constructs were introduced into E. coli strain BL21-CodonPlus (DE3)-RIPL (Stratagene).…”

“…Purified proteins were aliquoted, flash-frozen in liquid nitrogen, and stored at −80°C. E. coli RibA and RibD, and maize ZmRIBR, were expressed and purified as described [13]. …”

“…Intermediates 1 and 2 spontaneously anomerize [9,18]; the anomers of intermediate 1 were not resolved by the HPLC conditions used, and those of intermediate 2 were partially resolved. Intermediate 3 was synthesized as described [13]; its concentration was estimated at 1.5 mM. Intermediates 1, 2, and 3 were not further purified and were freshly prepared before use in assays.…”

“…Second, the first two intermediates are highly reactive and have half-lives of a few hours [9–11]. It is consequently suggestive that the third enzyme (RIBR) of riboflavin biosynthesis in plants [12,13] and homologs of the first enzyme (RibA) in bacteria such as Vibrio vulnificus are fused to a conserved domain of unknown function (COG3236 or DUF1768) that is almost surely an enzyme [14] (Figure 1B). Furthermore, a mutation ( phs1 ) that deletes the COG3236 domain of Arabidopsis RIBR is reported to reduce flavin content [12].…”