Over-expression of the yeast multifunctional arom protein.

The pentafunctional arom protein of Saccharomyces cerevisiae is encoded by the ARO1 gene. Substantial elevation of the levels of the arom protein (25-fold) was achieved in yeast using a vector that exploited the ubiquitin-fusion cleavage system of yeast. However, attempts to express the N-terminal 3-dehydroquinate synthase domain (E1) or the internal 3-dehydroquinase domain (E2) using the same system did not succeed. The yeast arom protein was successfully purified from the over-expressing transformant, and was found to possess all five enzymatic activities in a ratio similar to that observed in crude cell extracts. The purified material consisted mainly of a polypeptide that co-migrated in SDS-PAGE with intact arom proteins from other species.

Analysis of the structural stability of the multidomain enzyme flavocytochrome P-450 BM3.

The unfolding and refolding of flavocytochrome P-450 BM3 and its constituent haem and flavin domains have been analysed, using guanidinium chloride (GdnHCl) as a denaturant. Enzyme activities are lost at GdnHCl concentrations too low to cause major changes in secondary structure (0.1-0.5 M). The losses are primarily due to time-dependent FMN removal. Fluorescence and visible CD spectroscopies show that FMN dissociation is complete by 0.7 M GdnHCl, whereas FAD removal is complete by 1.5 M GdnHCl. Limited regain of activity is achieved by dilution of enzyme from solutions of < or = 0.75 M GdnHCl into fresh buffer. Supplementation of GdnHCl-free assay media with flavins (FAD and FMN) causes small additional regains in flavin domain (cytochrome-c reductase) activity lost at low [GdnHCl]. However, flavin addition during the denaturation step affords greater protection against inactivation, suggesting that conformational changes may occur subsequent to flavin loss and that these changes are not readily reversed on dilution of GdnHCl. Loss of catalytically competent haem ligation occurs over the same [GdnHCl] range for P-450 BM3 and its haem domain. In both cases, the 'denatured' P-420 form accumulates in the reduced/carbon monoxide-bound visible spectrum from 0.5 to 2 M GdnHCl. Secondary structure loss also occurs over similar [GdnHCl] ranges for P-450 BM3 and its two domains (80-90% lost from 0.5-3 M GdnHCl), indicating that there is little mutual stabilisation of domains in the holoenzyme. Differential scanning calorimetry measurements support this conclusion, but show that the haem domain is more thermostable than the flavin domain.

NADPH oxidase activity of cytochrome P-450 BM3 and its constituent reductase domain.

Cytochrome P-450 BM3 from Bacillus megaterium catalyses NADPH oxidation in the absence of added substrate. This activity is also associated with the independently expressed flavin-containing reductase domain of the protein. The rates of these activities are more than two orders of magnitude lower than those in the presence of fatty acid P-450 substrates or artificial electron acceptors. Electrons derived from NADPH in this fashion are transferred onto oxygen, generating superoxide (O2-) anions. The formation of these active oxygen species is detectable by luminometry and the chemiluminescence can be inhibited through the addition of superoxide dismutase (but not catalase). This activity is reminiscent of the microbicidal NADPH oxidase activity associated with neutrophils and other leukocyte blood cell types. Diphenyliodonium, a potent inhibitor of the neutrophil NADPH oxidase, effectively inhibits fatty acid hydroxylase and electron transferase activities catalysed by P-450 BM3 and its reductase domain. CD studies on the native and NADPH-reduced P-450 BM3 and BM3 reductase indicate that no secondary structural alteration is caused by pre-incubation with the reductant. Therefore, the previously recognised reversible time-dependent inactivation of P-450 BM3 by NADPH may be attributed to the NADPH oxidase activity associated with the reductase domain of the enzyme.

The unfolding and attempted refolding of the bacterial chaperone protein groEL (cpn60).

The unfolding of the bacterial chaperone protein groEL (cpn60) in solutions of guanidinium chloride (GdnHCl) has been studied. From the results of CD, fluorescence and light scattering, it is clear that major structural transitions in the protein occur over the range 1.0-1.5 M GdnHCl. The ATPase activity of the protein is lost at lower concentrations (0.75 M). After denaturation in concentrations of GdnHCl above 1.5 M, removal of the denaturing agent by dialysis results in very nearly complete regain of secondary structure (as judged by CD), but not the regain of correct tertiary or quaternary structure, or ATPase activity. The product was shown to be very sensitive to proteolysis by thermolysin, unlike the native protein, and not to show enhanced binding of ANS, a characteristic property of the 'molten globule' state of proteins. The results are discussed in relation to current information concerning the assembly of the groEL protein.

A eukaryotic repressor protein, the qa-1S gene product of Neurospora crassa, is homologous to part of the arom multifunctional enzyme.

Little is known about the proteins involved in the control of gene expression in eukaryotes. Although some of these proteins have been sequenced, their biochemical functions are not well understood and the identification of homologies to proteins of known activities may give useful clues to the functions of these regulatory proteins. We report here that the qa-1S repressor protein of the fungus Neurospora crassa is strongly homologous to the pentafunctional arom enzyme found in many lower eukaryotes. We believe that this is the first known case of a repressor protein that is unequivocally homologous to metabolic enzymes. These findings allow us to propose likely functions for some regions of the qa-1S protein.

The three-dimensional structure of shikimate kinase.

The three-dimensional structure of shikimate kinase from Erwinia chrysanthemi has been determined by multiple isomorphous replacement. Two models are presented: a high resolution 1.9 A model and a 2.6 A model which contains bound Mg-ADP. The enzyme is an alpha/beta protein consisting of a central sheet of five parallel beta-strands flanked by alpha-helices with overall topology similar to adenylate kinase. Evidence is presented that shikimate kinase undergoes major conformational changes on ligand binding. It resembles adenylate kinase in having a P-loop containing core structure and two flexible domains which undergo induced fit movement on substrate binding. The binding of Mg2+ in the active site of shikimate kinase involves direct interaction with two protein side-chains which is different from the situation found in adenylate kinase. Shikimate kinase has a readily identifiable Walker A-motif and a recognisable but modified Walker B-motif. Comparison of shikimate kinase to adenylate kinase has led to the identification of an adenine-binding motif (I/VDAXQ/NXP). Difference Fourier calculations have revealed the shikimate binding site which corresponds to the location of the AMP-binding site in adenylate kinase. A model for shikimate-binding is presented.

Evidence for an ancestral core structure in nucleotide-binding proteins with the type A motif.

Many proteins that bind purine nucleotide triphosphates have a type A sequence motif. Only two classes of structures for such proteins are so far available from X-ray crystallography. We examined the tertiary structures of representatives of the two classes, porcine cytoplasmic adenylate kinase and Escherichia coli translational elongation factor Tu. Comparison of the two proteins suggests that the A motif may be just one part of a larger common core structure consisting of four parallel strands of beta-sheet sandwiched between four alpha-helices. This compact core structure comprises over one half of each protein. We speculate that A motif proteins have diverged from a common ancestor having this core structure.

Crystallization of a type II dehydroquinase from Mycobacterium tuberculosis.

A type II 3-dehydroquinase from Mycobacterium tuberculosis has been crystallized in the presence of 6% polyethyleneglycol 6000. Data from these crystals have been collected to a resolution of 2.2 A on a rotating anode X-ray source. The space group has been determined as F23 with unit cell dimensions of a = b = c = 127.8 A. There is one molecule in the asymmetric unit.

Crystallization of a type I 3-dehydroquinase from Salmonella typhi.

Crystals have been grown of a type I 3-dehydroquinase from both Escherichia coli and Salmonella typhi. However, only those from S. typhi diffract to a resolution of 2.3 A on a conventional X-ray source and are suitable for structure determination. The space group has been determined as P2(1)2(1)2 with unit cell dimensions a = 48.01 A, b = 114.29 A, c = 42.87 A. There is one subunit in the asymmetric unit.

The efficiency of folding of some proteins is increased by controlled rates of translation in vivo. A hypothesis.

We propose that the way in which some proteins fold is affected by the rates at which regions of their polypeptide chains are translated in vivo. Furthermore, we suggest that their gene sequences have evolved to control the rate of translational elongation such that the synthesis of defined portions of their polypeptide chains is separated temporally. We stress that many proteins are capable of folding efficiently into their native conformations without the help of differential translation rates. For these proteins the amino acid sequence does indeed contain all the information needed for the polypeptide chain to fold correctly (even in vitro, after denaturation). However, other proteins clearly do not fold efficiently into their native conformation in vitro. We argue that the efficiency of folding of these problematic proteins in vivo may be improved by controlled synthesis of the nascent polypeptide.

Deletions, fusions and domain rearrangements.

The techniques of protein engineering are proving to be powerful analytical tools for the study of the structure and function of complex multidomain proteins. In particular, the overexpression of individual functional modules is providing proteins for three-dimensional structural analyses. Progress is also being made in the design and construction of novel multidomain proteins with potential therapeutic applications.

Biochemical and X-ray crystallographic studies on shikimate kinase: the important structural role of the P-loop lysine.

Shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase. In this paper we have explored the roles of residues in the P-loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins. In common with many members of the P-loop family, shikimate kinase contains a cysteine residue 2 amino acids upstream of the essential lysine residue; the side chains of these residues are shown to form an ion pair. The C13S mutant of shikimate kinase was found to be enzymatically active, whereas the K15M mutant was inactive. However, the latter mutant had both increased thermostability and affinity for ATP when compared to the wild-type enzyme. The structure of the K15M mutant protein has been determined at 1.8 A, and shows that the organization of the P-loop and flanking regions is heavily disturbed. This indicates that, besides its role in catalysis, the P-loop lysine also has an important structural role. The structure of the K15M mutant also reveals that the formation of an additional arginine/aspartate ion pair is the most likely reason for its increased thermostability. From studies of ligand binding it appears that, like adenylate kinase, shikimate kinase binds substrates randomly and in a synergistic fashion, indicating that the two enzymes have similar catalytic mechanisms.

The complete amino acid sequence of 3-dehydroquinate synthase of Escherichia coli K12.

The complete amino acid sequence of the Escherichia coli 3-dehydroquinate synthase has been determined by a combined nucleotide and direct amino acid sequencing strategy. E. coli 3-dehydroquinate synthase is 362 amino acids long and has a calculated Mr of 38 880. Analysis of the aroB nucleotide sequence and its 5'- and 3'-flanking regions has identified the aroB promoter elements and a possible 3'-terminator site.

Kinetics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate.

The herbicide glyphosate (N-phosphonomethyl glycine) is a potent reversible inhibitor of the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase activity of the purified arom multienzyme complex from Neurospora crassa. Inhibition of the EPSP synthase reaction by glyphosate is competitive with respect to phosphoenolpyruvate, with K(i) 1.1 microM, and uncompetitive with respect to shikimate-3-phosphate. The kinetic patterns are consistent with a compulsory order sequential mechanism in which either PEP or glyphosate can bind to an enzyme: shikimate-3-phosphate complex.

The Saccharomyces cerevisiae ARO1 gene. An example of the co-ordinate regulation of five enzymes on a single biosynthetic pathway.

The ARO1 gene of Saccharomyces cerevisiae encodes the arom multifunctional enzyme. Specific inhibitors of amino acid biosynthesis have been used to obtain evidence that expression of a cloned ARO1 gene is regulated in response to amino acid limitation. Northern blot analysis and sequence studies indicate that ARO1 is regulated by the well characterised S. cerevisiae 'general control' mechanism. This provides a very economical means of simultaneously tailoring the synthesis of five shikimate pathway enzymes to the needs of the cell.

The use of electrospray mass spectrometry to identify an essential arginine residue in type II dehydroquinases.

The arginine-specific reagent phenylglyoxal has been used to identify a hyper-reactive arginine residue which is essential for activity in the type II dehydroquinases of Streptomyces coelicolor and Aspergillus nidulans. Electrospray mass spectrometry was used both to characterise the phenylglyoxal modified protein, and to identify the phenylglyoxal modified peptides following enzymatic digestion. The advantages of using electrospray mass spectrometry for monitoring arginine modication aimed at identifying functional residues in proteins are discussed.

Evidence from peptide mapping for a human renin zymogen.

Data obtained from peptide mapping of the active and inactive forms of human renin show that there are extensive regions of common sequence in the two forms of the enzyme, and are consistent with the hypothesis that inactive renin is a renin zymogen.

The purification of 5-enolpyruvylshikimate 3-phosphate synthase from an overproducing strain of Escherichia coli.

The Escherichia coli aroA gene which codes for the enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSP synthase) has been cloned from the lambda-transducing bacteriophage lambda pserC. The gene has been located on a 4.7 kilobase pair PstI DNA fragment which has been inserted into the multiple copy plasmid pAT153. E. coli cells transformed with this recombinant plasmid overproduce EPSP synthase 100-fold. A simple method for the purification of homogeneous enzyme in milligram quantities has been devised. The resulting enzyme is indistinguishable from enzyme isolated from untransformed E. coli.

The characterisation of the shikimate pathway enzyme dehydroquinase from Pisum sativum.

Peptides accounting for 157 residues of the bifunctional shikimate pathway enzyme, dehydroquinase/shikimate dehydrogenase, of Pisum sativum were sequenced. Three of the peptides were homologous to regions in Escherichia coli dehydroquinase and two to E. coli shikimate dehydrogenase. The pea dehydroquinase activity was inhibited by treatment with dehydroquinate plus sodium borohydride, establishing it as a type I dehydroquinase. Synthetic oligonucleotides designed from the amino acid sequence were used as PCR primers to amplify fragments of P. sativum cDNA. DNA sequence analysis showed that these amplified products were derived from dehydroquinase/shikimate dehydrogenase cDNA. The complete amino acid sequence of the dehydroquinase domain has been defined; it is homologous to all other type I dehydroquinases and is N-terminal.

The reaction of GroEL (cpn 60) with the ATP analogue 2',3' dialdehyde ATP.

The reaction of the E. coli chaperonin GroEL (cpn 60) with the ATP analogue 2',3' oxidised ATP (oATP) has been studied. Treatment with the reagent leads to loss of the ATPase activity of GroEL in a pseudo-first-order fashion; this can be prevented by inclusion of ATP in the reaction mixture. Measurements of the stoichiometry of the reaction indicate that the loss of activity corresponds to the incorporation of about one oATP per subunit of GroEL. From analysis of the sequences of modified peptides it is proposed that the reaction probably occurs with one or both of the two cysteines Cys-457 and Cys-518, although the instability of the adduct(s) makes a definite identification of the site(s) of reaction difficult. The involvement of Cys side chains in the reaction with oATP was confirmed by using Nbs2 (5,5'-dithiobis(2-nitrobenzoate)) to estimate thiol groups in both modified and unmodified GroEL.