Crystal structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase of riboflavin biosynthesis.

BACKGROUND: 3,4-Dihydroxy-2-butanone-4-phosphate synthase catalyzes a commitment step in the biosynthesis of riboflavin. On the enzyme, ribulose 5-phosphate is converted to 3,4-dihydroxy-2-butanone 4-phosphate and formate in steps involving enolization, ketonization, dehydration, skeleton rearrangement, and formate elimination. The enzyme is absent in humans and an attractive target for the discovery of antimicrobials for pathogens incapable of acquiring sufficient riboflavin from their hosts. The homodimer of 23 kDa subunits requires Mg(2+) for activity. RESULTS: The first three-dimensional structure of the enzyme was determined at 1.4 A resolution using the multiwavelength anomalous diffraction (MAD) method on Escherichia coli protein crystals containing gold. The protein consists of an alpha + beta fold having a complex linkage of beta strands. Intersubunit contacts are mediated by numerous hydrophobic interactions and three hydrogen bond networks. CONCLUSIONS: A proposed active site was identified on the basis of amino acid residues that are conserved among the enzyme from 19 species. There are two well-separated active sites per dimer, each of which comprise residues from both subunits. In addition to three arginines and two threonines, which may be used for recognizing the phosphate group of the substrate, the active site consists of three glutamates, two aspartates, two histidines, and a cysteine which may provide the means for general acid and base catalysis and for coordinating the Mg(2+) cofactor within the active site.

Crystal structure of riboflavin synthase.

BACKGROUND: Riboflavin synthase catalyzes the dismutation of two molecules of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine to yield riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine. The homotrimer of 23 kDa subunits has no cofactor requirements for catalysis. The enzyme is nonexistent in humans and is an attractive target for antimicrobial agents of organisms whose pathogenicity depends on their ability to biosynthesize riboflavin. RESULTS: The first three-dimensional structure of the enzyme was determined at 2.0 A resolution using the multiwavelength anomalous diffraction (MAD) method on the Escherichia coli protein containing selenomethionine residues. The homotrimer consists of an asymmetric assembly of monomers, each of which comprises two similar beta barrels and a C-terminal alpha helix. The similar beta barrels within the monomer confirm a prediction of pseudo two-fold symmetry that is inferred from the sequence similarity between the two halves of the protein. The beta barrels closely resemble folds found in phthalate dioxygenase reductase and other flavoproteins. CONCLUSIONS: The three active sites of the trimer are proposed to lie between pairs of monomers in which residues conserved among species reside, including two Asp-His-Ser triads and dyads of Cys-Ser and His-Thr. The proposed active sites are located where FMN (an analog of riboflavin) is modeled from an overlay of the beta barrels of phthalate dioxygenase reductase and riboflavin synthase. In the trimer, one active site is formed, and the other two active sites are wide open and exposed to solvent. The nature of the trimer configuration suggests that only one active site can be formed and be catalytically competent at a time.

Cloning, expression, purification and crystallization of dihydroxybutanone phosphate synthase from Magnaporthe grisea.

Dihydroxybutanone phosphate synthase (DS) catalyzes a commitment step in riboflavin biosynthesis where ribulose 5-phosphate is converted to dihydroxybutanone phosphate and formate. DS was cloned from the pathogenic fungus Magnaporthe grisea (using functional complementation of an Escherichia coli DS knockout mutant) and expressed in E. coli. The purified protein crystallized in space group P2(1)2(1)2. Diffraction data extending to 1.5, 1.0 and 1.8 A resolution were collected from crystals that were divalent cation free, soaked in Zn(2+) or soaked in Mg(2+), respectively.

Cloning of the SNG1 gene of Arabidopsis reveals a role for a serine carboxypeptidase-like protein as an acyltransferase in secondary metabolism.

Serine carboxypeptidases contain a conserved catalytic triad of serine, histidine, and aspartic acid active-site residues. These enzymes cleave the peptide bond between the penultimate and C-terminal amino acid residues of their protein or peptide substrates. The Arabidopsis Genome Initiative has revealed that the Arabidopsis genome encodes numerous proteins with homology to serine carboxypeptidases. Although many of these proteins may be involved in protein turnover or processing, the role of virtually all of these serine carboxypeptidase-like (SCPL) proteins in plant metabolism is unknown. We previously identified an Arabidopsis mutant, sng1 (sinapoylglucose accumulator 1), that is defective in synthesis of sinapoylmalate, one of the major phenylpropanoid secondary metabolites accumulated by Arabidopsis and some other members of the Brassicaceae. We have cloned the gene that is defective in sng1 and have found that it encodes a SCPL protein. Expression of SNG1 in Escherichia coli demonstrates that it encodes sinapoylglucose:malate sinapoyltransferase, an enzyme that catalyzes a transesterification instead of functioning like a hydrolase, as do the other carboxypeptidases. This finding suggests that SCPL proteins have acquired novel functions in plant metabolism and provides an insight into the evolution of secondary metabolic pathways in plants.

Reconstitution of higher plant chloroplast chaperonin 60 tetradecamers active in protein folding.

Unlike the GroEL homologs of eubacteria and mitochondria, oligomer preparations of the higher plant chloroplast chaperonin 60 (cpn60) consist of roughly equal amounts of two divergent subunits, alpha and beta. The functional significance of these isoforms, their structural organization into tetradecamers, and their interactions with the unique binary chloroplast chaperonin 10 (cpn10) have not been elucidated. Toward this goal, we have cloned the alpha and beta subunits of the ch-cpn60 of pea (Pisum sativum), expressed them individually in Escherichia coli, and subjected the purified monomers to in vitro reconstitution experiments. In the absence of other factors, neither subunit (alone or in combination) spontaneously assembles into a higher order structure. However, in the presence of MgATP, the beta subunits form tetradecamers in a cooperative reaction that is potentiated by cpn10. In contrast, alpha subunits only assemble in the presence of beta subunits. Although beta and alpha/beta 14-mers are indistinguishable by electron microscopy and can both assist protein folding, their specificities for cpn10 are entirely different. Similar to the authentic chloroplast protein, the reconstituted alpha/beta 14-mers are functionally compatible with bacterial, mitochondrial, and chloroplast cpn10. In contrast, the folding reaction mediated by the reconstituted beta 14-mers is only efficient with mitochondrial cpn10. The ability to reconstitute two types of functional oligomer in vitro provides a unique tool, which will allow us to investigate the mechanism of this unusual chaperonin system.

Crystal structure analysis of a pentameric fungal and an icosahedral plant lumazine synthase reveals the structural basis for differences in assembly.

Lumazine synthase catalyzes the penultimate step in the synthesis of riboflavin in plants, fungi, and microorganisms. The enzyme displays two quaternary structures, the pentameric forms in yeast and fungi and the 60-meric icosahedral capsids in plants and bacteria. To elucidate the structural features that might be responsible for differences in assembly, we have determined the crystal structures of lumazine synthase, complexed with the inhibitor 5-nitroso-6-ribitylamino-2,4-pyrimidinedione, from spinach and the fungus Magnaporthe grisea to 3.3 and 3.1 A resolution, respectively. The overall structure of the subunit and the mode of inhibitor binding are very similar in these enzyme species. The core of the subunit consists of a four-stranded parallel beta-sheet sandwiched between two helices on one side and three helices on the other. The packing of the five subunits in the pentameric M. grisea lumazine synthase is very similar to the packing in the pentameric substructures in the icosahedral capsid of the plant enzyme. Two structural features can be correlated to the differences in assembly. In the plant enzyme, the N-terminal beta-strand interacts with the beta-sheet of the adjacent subunit, thus extending the sheet from four to five strands. In fungal lumazine synthase, an insertion of two residues after strand beta1 results in a completely different orientation of this part of the polypeptide chain and this conformational difference prevents proper packing of the subunits at the trimer interface in the icosahedron. In the spinach enzyme, the beta-hairpin connecting helices alpha4 and alpha5 participates in the packing at the trimer interface of the icosahedron. Another insertion of two residues at this position of the polypeptide chain in the fungal enzyme disrupts the hydrogen bonding in the hairpin, and the resulting change in conformation of this loop also interferes with proper intrasubunit contacts at the trimer interface.

Plant riboflavin biosynthesis. Cloning, chloroplast localization, expression, purification, and partial characterization of spinach lumazine synthase.

Lumazine synthase, which catalyzes the penultimate step of riboflavin biosynthesis, has been cloned from three higher plants (spinach, tobacco, and arabidopsis) through functional complementation of an Escherichia coli auxotroph. Whereas the three plant proteins exhibit some structural similarities to known microbial homologs, they uniquely possess N-terminal polypeptide extensions that resemble typical chloroplast transit peptides. In vitro protein import assays with intact chloroplasts and immunolocalization experiments verify that higher plant lumazine synthase is synthesized in the cytosol as a larger molecular weight precursor protein, which is post-translationally imported into chloroplasts where it is proteolytically cleaved to its mature size. The authentic spinach enzyme is estimated to constitute <0.02% of the total chloroplast protein. Recombinant "mature" spinach lumazine synthase is expressed in E. coli at levels exceeding 30% of the total soluble protein and is readily purified to homogeneity using a simple two-step procedure. Apparent V(max) and K(m) values obtained with the purified plant protein are similar to those reported for microbial lumazine synthases. Electron microscopy and hydrodynamic studies reveal that native plant lumazine synthase is a hollow capsid-like structure comprised of 60 identical 16.5-kDa subunits, resembling its icosahedral counterparts in E. coli and Bacillus subtilis.

Chaperonin function depends on structure and disorder in co-chaperonin mobile loops.

Co-chaperonins from diverse organisms exhibit mobile loops which fold into a beta hairpin conformation upon binding to the chaperonin. GroES, Gp31, and human Hsp10 mobile loops exhibit a preference for the beta hairpin conformation in the free co-chaperonins, and the conformational dynamics of the human Hsp10 mobile loop appear to be restricted by nascent hairpin formation. Backbone conformational entropy must weigh against binding of co-chaperonins to chaperonins, and thus the conformational preferences of the loops may strongly influence chaperonin-binding affinity. Indeed, subtle mutations in the loops change GroEL-binding affinity and cause defects in chaperonin function, and these defects can be suppressed by mutations in GroEL which compensate for the changes in affinity. The fact that high-affinity co-chaperonin binding impairs chaperonin function has implications for the mechanism of chaperonin-assisted protein folding.

Molecular chaperones and protein folding in plants.

Protein folding in vivo is mediated by an array of proteins that act either as 'foldases' or 'molecular chaperones'. Foldases include protein disulfide isomerase and peptidyl prolyl isomerase, which catalyze the rearrangement of disulfide bonds or isomerization of peptide bonds around Pro residues, respectively. Molecular chaperones are a diverse group of proteins, but they share the property that they bind substrate proteins that are in unstable, non-native structural states. The best understood chaperone systems are HSP70/DnaK and HSP60/GroE, but considerable data support a chaperone role for other proteins, including HSP100, HSP90, small HSPs and calnexin. Recent research indicates that many, if not all, cellular proteins interact with chaperones and/or foldases during their lifetime in the cell. Different chaperone and foldase systems are required for synthesis, targeting, maturation and degradation of proteins in all cellular compartments. Thus, these diverse proteins affect an exceptionally broad array of cellular processes required for both normal cell function and survival of stress conditions. This review summarizes our current understanding of how these proteins function in plants, with a major focus on those systems where the most detailed mechanistic data are available, or where features of the chaperone/foldase system or substrate proteins are unique to plants.

Functional characterization of the higher plant chloroplast chaperonins.

The higher plant chloroplast chaperonins (ch-cpn60 and ch-cpn10) have been purified and their structural/functional properties examined. In all plants surveyed, both proteins were constitutively expressed, and only modest increases in their levels were detected upon heat shock. Like GroEL and GroES of Escherichia coli, the chloroplast chaperonins can physically interact with each other. The asymmetric complexes that form in the presence of ADP are "bullet-shaped" particles that likely consist of 1 mol each of ch-cpn60 and ch-cpn10. The purified ch-cpn60 is a functional molecular chaperone. Under "nonpermissive" conditions, where spontaneous folding was not observed, it was able to assist in the refolding of two different target proteins. In both cases, successful partitioning to the native state also required ATP hydrolysis and chaperonin 10. Surprisingly, however, the "double-domain" ch-cpn10, comprised of unique 21-kDa subunits, was not an obligatory co-chaperonin. Both GroES and a mammalian mitochondrial homolog were equally compatible with the ch-cpn60. Finally, the assisted-folding reaction mediated by the chloroplast chaperonins does not require K+ ions. Thus, the K(+)-dependent ATPase activity that is observed with other known groEL homologs is not a universal property of all chaperonin 60s.

Cloning, expression, and purification of a functional nonacetylated mammalian mitochondrial chaperonin 10.

An intact mouse mitochondrial chaperonin 10 has been cloned, sequenced, and overexpressed in Escherichia coli as a fusion protein harboring an oligohistidine tail at its COOH terminus. The latter was added to simplify protein purification. The purified protein is free of contaminating groES from the bacterial host cells. Edman degradation reveals that the initiator Met residue of the recombinant protein is removed in vivo, similar to the authentic chaperonin 10 purified from rat liver mitochondria. However, in contrast to the latter, the amino-terminal Ala residue of the recombinant protein is not acetylated; the molecular mass determined by electrospray ionization mass spectrometry is 12,350.9 +/- 2.6 daltons, in agreement with that predicted for the nonacetylated protein (12,351.2 daltons). Facilitated protein folding experiments with ribulose-biphosphate carboxylase, under "nonpermissive" in vitro conditions, demonstrate that the recombinant protein is fully functional with groEL. Thus, both the initial rates of protein folding and final yields observed with this heterologous combination are virtually identical to those obtained with groEL and groES. More important, like the authentic protein purified from mitochondria, the recombinant mitochondrial chaperonin 10, but not groES, is functionally compatible with the heptameric chaperonin 60 of mammalian mitochondria.

Dynamics of the chaperonin ATPase cycle: implications for facilitated protein folding.

The Escherichia coli chaperonins GroEL and GroES facilitate protein folding in an adenosine triphosphate (ATP)-dependent manner. After a single cycle of ATP hydrolysis by the adenosine triphosphatase (ATPase) activity of GroEL, the bi-toroidal GroEL formed a stable asymmetric ternary complex with GroES and nucleotide (bulletlike structures). With each subsequent turnover, ATP was hydrolyzed by one ring of GroEL in a quantized manner, completely releasing the adenosine diphosphate and GroES that were tightly bound to the other ring as a result of the previous turnover. The catalytic cycle involved formation of a symmetric complex (football-like structures) as an intermediate that accumulated before the rate-determining hydrolytic step. After one to two cycles, most of the substrate protein dissociated still in a nonnative state, which is consistent with intermolecular transfer of the substrate protein between toroids of high and low affinity. A unifying model for chaperonin-facilitated protein folding based on successive rounds of binding and release, and partitioning between committed and kinetically trapped intermediates, is proposed.

On the role of groES in the chaperonin-assisted folding reaction. Three case studies.

The mechanism by which correctly folded proteins are recovered from stable complexes with groEL is not well understood. Certain target proteins require ATP and groES, while others seemingly dispense with the cochaperonin. Here, we examine the chaperonin-assisted folding of ribulose-1,5-bisphosphate carboxylase, malate dehydrogenase, and citrate synthase, three proteins that are believed to require both chaperonin components for successful reactivation. Surprisingly, in all cases, the need for groES depended on the folding environment. Under "non-permissive" conditions, where unassisted spontaneous folding could not occur, reactivation to the native state required the complete chaperonin system (e.g. groEL, groES, and MgATP). However, under "permissive" conditions where spontaneous folding could occur groES was no longer mandatory. Instead, upon the addition of ATP alone, all three target proteins could be released from groEL, in a form that was capable of reaching the native state. In the permissive setting, groES merely accelerated the rate of the ATP-dependent release process. The results suggest that the incompletely folded protein species that are released from groEL, in the absence of groES, are not necessarily committed to the native state. Similar to the unassisted folding reaction, they still partition between productive and unproductive folding pathways in an environment-dependent manner. It follows that the mechanistic contribution of the co-chaperonin, groES, and its physiological significance in cellular protein folding, could be entirely missed in a permissive in vitro environment.

Identification and functional analysis of chaperonin 10, the groES homolog from yeast mitochondria.

Chaperonin 60 (cpn60) and chaperonin 10 (cpn10) constitute the chaperonin system in prokaryotes, mitochondria, and chloroplasts. In Escherichia coli, these two chaperonins are also termed groEL and groES. We have used a functional assay to identify the groES homolog cpn10 in yeast mitochondria. When dimeric ribulose-1,5-bisphosphate carboxylase (Rubisco) is denatured and allowed to bind to yeast cpn60, subsequent refolding of Rubisco is strictly dependent upon yeast cpn10. The heterologous combination of cpn60 from E. coli plus yeast cpn10 is also functional. In contrast, yeast cpn60 plus E. coli cpn10 do not support refolding of Rubisco. In the presence of MgATP, yeast cpn60 and yeast cpn10 form a stable complex that can be isolated by gel filtration and that facilitates refolding of denatured Rubisco. Although the potassium-dependent ATPase activity of E. coli cpn60 can be inhibited by cpn10 from either E. coli or yeast, neither of these cpn10s inhibits the ATPase activity of yeast cpn60. Amino acid sequencing of yeast cpn10 reveals substantial similarity to the corresponding cpn10 proteins from rat mitochondria and prokaryotes.

Hydrolysis of adenosine 5'-triphosphate by Escherichia coli GroEL: effects of GroES and potassium ion.

The potassium-ion activation constant (Kact) for the ATPase activity of Escherichia coli chaperonin groEL is inversely dependent upon the ATP concentration over at least 3 orders of magnitude. The ATPase activity shows positively cooperative kinetics with respect to ATP and K+. Both the K0.5 for ATP and cooperativity (as measured by the Hill coefficient) decrease as the K+ concentration increases. Equilibrium binding studies under conditions where hydrolysis does not occur indicate that MgATP binds weakly to groEL in the absence of K+. In the absence of groES, the K(+)-dependent hydrolysis of ATP by groEL continues to completion. In the presence of groES, the time course for the hydrolysis of ATP by groEL becomes more complex. Three distinct kinetic phases can be discerned. Initially, both heptameric toroids turn over once at the same rate that they do in the absence of groES. This leads to the formation of an asymmetric binary complex, groEL14-MgADP7-groES7, in which 7 mol of ADP is trapped in a form that does not readily exchange with free ADP. In the second phase, the remaining seven sites (containing readily exchangeable ADP) turn over, or have the potential to turn over, at the same rate as they do in the absence of groES, so that the overall rate of hydrolysis is maximally 50%. These remaining sites of the asymmetric binary complex do not hydrolyze all of the available ATP. Instead, the second phase of hydrolysis gives way to a third, completely inhibited state, the onset of which is dependent upon the relative affinities of the remaining sites for MgATP and MgADP.(ABSTRACT TRUNCATED AT 250 WORDS)

Chaperonins and protein folding: unity and disunity of mechanisms.

Chaperonin-facilitated folding of proteins involves two partial reactions. The first partial reaction, the formation of stable binary complexes between chaperonin-60 and non-native states of the target protein, is common to the chaperonin-facilitated folding of all target proteins investigated to date. The structural basis for this interaction is not presently understood. The second partial reaction, the dissociation of the target protein in a form committed to the native state, appears to proceed by a variety of mechanisms, dependent upon the nature of the target protein in question. Those target proteins (e.g. rubisco, rhodanese, citrate synthase) which require the presence of chaperonin-10, also appear to require the hydrolysis of ATP to bring about the dissociation of the target protein from chaperonin-60. With one exception (pre-beta-lactamase) those target proteins which do not require the presence of chaperonin-10 to be released from chaperonin-60, also do not require the hydrolysis of ATP, since non-hydrolysable analogues of ATP support the release of the target protein in a state committed to the native state. The question of whether or not chaperonin-facilitated folding constitutes a catalysed event is addressed.

Identification, characterization, and DNA sequence of a functional "double" groES-like chaperonin from chloroplasts of higher plants.

Chloroplasts of higher plants contain a nuclear-encoded protein that is a functional homolog of the Escherichia coli chaperonin 10 (cpn10; also known as groES). In pea (Pisum sativum), chloroplast cpn10 was identified by its ability to (i) assist bacterial chaperonin 60 (cpn60; also known as groEL) in the ATP-dependent refolding of chemically denatured ribulose-1,5-bisphosphate carboxylase and (ii) form a stable complex with bacterial cpn60 in the presence of Mg.ATP. The subunit size of the pea protein is approximately 24 kDa--about twice the size of bacterial cpn10. A cDNA encoding a spinach (Spinacea oleracea) chloroplast cpn10 was isolated, sequenced, and expressed in vitro. The spinach protein is synthesized as a higher molecular mass precursor and has a typical chloroplast transit peptide. Surprisingly, however, attached to the transit peptide is a single protein, comprised of two distinct cpn10 molecules in tandem. Moreover, both halves of this "double" cpn10 are highly conserved at a number of residues that are present in all cpn10s that have been examined. Upon import into chloroplasts the spinach cpn10 precursor is processed to its mature form of approximately 24 kDa. N-terminal amino acid sequence analysis reveals that the mature pea and spinach cpn10 are identical at 13 of 21 residues.