Structural Analysis of Escherichia Coli ThiF

Escherichia coli ThiF is an enzyme in the biosynthetic cascade for generating the essential cofactor thiamin pyrophosphate. In this cascade, ThiF catalyzes adenylation of the C terminus of ThiS. We report here the crystal structures of ThiF, alone and in complex with ATP. The structures provide insight into a preference for ATP during adenylation of the protein ThiS. Additionally, the structures reveal an ordered crossover loop predicted to clamp the flexible tail of ThiS into the ThiF active site during the adenylation reaction. The importance of the crossover loop for ThiF activity is highlighted by mutational analysis. Comparison of ThiF with the structural homologues MoeB, APPBP1-UBA3, and SAE1-SAE2 reveals that the ATP-binding site, including an arginine-finger, is maintained throughout evolution, and shows divergence occurring in protein substrate-binding sites and regions devoted to unique steps in the specific function of each enzyme.

Introduction

Thiamin pyrophosphate is an essential cofactor in carbohydrate and branched amino acid metabolism. The biosynthesis of thiamin involves a complex cascade of enzymes with distinct paths for synthesizing the thiazole and pyrimidine moieties, which are then coupled and phosphorylated to yield thiamin pyrophosphate.1, 2, 3, 4 In Escherichia coli, formation of the thiazole moiety involves the actions of six distinct gene products: ThiS, ThiF, ThiG, ThiH, IscS and ThiI.5, 6, 7 The thiazole moiety is synthesized from deoxy-d-xylulose 5-phosphate (DXP), tyrosine, and a sulfur atom carried by the C terminus of ThiS, a 66 residue protein (Figure 1(a)).

ThiF is an essential enzyme for thiamin biosynthesis.5, 8 Current evidence suggests that ThiF is involved in two distinct steps in the generation of the thiazole moiety in E. coli. First, ThiF catalyzes formation of a critical ThiS-acyl-adenylate (ThiS-COAMP) intermediate. Evidence for ThiF-catalyzed adenylation at the ThiS C terminus is as follows: (1) a purified ThiF–ThiS complex was shown to catalyze formation of pyrophosphate in a MgATP-dependent manner; and (2) mass spectrometric characterization of MgATP-treated ThiF–ThiS purified from a ThiI⁻ strain of E. coli revealed the presence of the ThiS-COAMP product.⁸ Following formation of ThiS-COAMP, IscS catalyzes transfer of sulfur from cysteine to yield ThiS-thiocarboxylate. ThiS-thiocarboxylate formation has been achieved in vitro in the presence of ThiF and IscS.6, 9 ThiI has been implicated in formation of the ThiS-thiocarboxylate intermediate, although ThiI is not essential, and the exact catalytic function of ThiI remains poorly understood.⁸ Subsequent to formation of the ThiS-thiocarboxylate, E. coli ThiF has been implicated in a second role in thiazole biosynthesis: a covalently linked ThiF–ThiS complex was identified from a partially purified mixture of ThiF and ThiS.¹⁰ Mass spectrometry revealed this complex contains an acyldisulfide linkage between the sulfur atoms of the ThiS-thiocarboxylate and Cys184 of ThiF.¹⁰ Mutation of ThiF Cys184 to serine did not affect generation of the ThiS-thiocarboxylate intermediate, indicating that formation of the acyldisulfide-linked ThiF–ThiS complex follows ThiS-thiocarboxylate formation.¹⁰ Although a covalently linked ThiF–ThiS complex has not been detected from Bacillus subtilis,¹¹ the physiological importance of the ThiF–ThiS covalent complex in E. coli is underscored by the finding that the ThiF Cys184Ser mutant did not complement the growth defect of ThiF⁻ E. coli.¹⁰ Exogenous addition of thiazole restores normal E. coli growth, attributing the defect to thiazole biosynthesis.¹⁰ Thus, previous results suggest two critical functions for ThiF in thiazole biosynthesis in E. coli: adenylation of the ThiS C terminus, and formation of a covalent acyldisulfide complex.¹⁰

Ultimately, the thiazole moiety is generated from DXP, tyrosine, and the sulfur atom carried at the C terminus of ThiS, in a reaction catalyzed by ThiG, or thiazole synthase.4, 11, 12 Interestingly, ThiG has been shown to compete with ThiF for binding to ThiS, indicating a requirement for the ThiF-mediated and ThiG-mediated reactions to occur sequentially.¹³

The structures of E. coli and B. subtilis ThiS have been determined recently, both alone in solution (from E. coli), and in complex with ThiG (from B. subtilis).13, 14 The ThiS structure consists of a globular domain containing a four-stranded β-sheet and an α-helix, and a flexible C-terminal tail that terminates in the sequence Gly–Gly. Interestingly, the ThiS structure revealed striking similarity to the E. coli protein MoaD, which also carries a sulfur atom at its C terminus for the biosynthesis of molybdopterin.¹⁵ In addition, ThiS resembles the eukaryotic protein ubiquitin, and ubiquitin-like proteins (ubls) such as NEDD8, SUMO and ISG15, raising the possibility of common ancestry for ThiS and ubiquitin and ubls.¹⁴ Ubiquitin and ubls are small proteins with C termini that become covalently attached to other proteins as post-translational modifications in eukaryotes.¹⁶ Ubiquitin and ubls are involved in essential eukaryotic mechanisms for regulating processes such as cell division, development, the immune system and cellular trafficking.16, 17 These post-translational modifications serve as tags that alter the activities of their targets, such as changing a protein's half-life, subcellular localization, or catalytic activity.18, 19, 20, 21, 22

Further evidence for an evolutionary relationship between the thiamin biosynthetic pathway in prokaryotes and ubiquitin and ubl post-translational modification pathways in eukaryotes comes from sequence similarity between ThiF and portions of the E1 enzymes that initiate ubiquitin and ubl conjugation.¹⁷ Ubiquitin and ubls are conjugated to their protein targets through parallel, multienzyme cascades.17, 23 For each ubl, a dedicated E1 catalyzes multiple reactions. First, the E1 binds ATP, Mg²⁺, and its specific ubl, and catalyzes adenylation of the ubl's C terminus. The E1 subsequently forms a covalent thioester intermediate complex between its catalytic cysteine residue and the C terminus of the ubl. The sequential E1-mediated adenylation and covalent thioester complex formation at the C terminus of ubiquitin and ubls is reminiscent of the ThiF-mediated adenylation and covalent acyldisulfide complex formation with ThiS (Figure 1(b)).8, 10 The ThiF-catalyzed adenylation of the ThiS C terminus also resembles the adenylation reaction catalyzed at the C terminus of E. coli MoaD by MoeB, which is essential for molybdopterin biosynthesis.24, 25 ThiF and MoeB share 44% sequence identity, and are likely evolutionary ancestors of the eukaryotic E1s, with both including a Gly-X-Gly-X-X-Gly nucleotide-binding motif.²⁶

In order to better understand the function of ThiF, we examined the nucleotide specificity of ThiF-catalyzed modification of the ThiS C terminus, and present here data supporting a strong preference for ATP over GTP, CTP or TTP. In order to gain insight into the mechanism of ThiF, we determined the crystal structures of ThiF, alone and in complex with ATP. The structures reveal similarities and differences between ThiF, MoeB, and APPBP1-UBA3 and SAE1-SAE2, the E1s for the ubls NEDD8 and SUMO, respectively.25, 27, 28 The structures shed light on the function of ThiF, and on the evolution of the common ThiF/MoeB/E1 fold.