Stanley E. Mills

The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, l-tryptophan

The crystal structure of anthranilate synthase (AS) from Serratia marcescens, a mesophilic bacterium, has been solved in the presence of its substrates, chorismate and glutamine, and one product, glutamate, at 1.95 Å, and with its bound feedback inhibitor, tryptophan, at 2.4 Å. In comparison with the AS structure from the hyperthermophile Sulfolobus solfataricus, the S. marcescens structure shows similar subunit structures but a markedly different oligomeric organization. One crystal form of the S. marcescens enzyme displays a bound pyruvate as well as a putative anthranilate (the nitrogen group is ambiguous) in the TrpE subunit. It also confirms the presence of a covalently bound glutamyl thioester intermediate in the TrpG subunit. The tryptophan-bound form reveals that the inhibitor binds at a site distinct from that of the substrate, chorismate. Bound tryptophan appears to prevent chorismate binding by a demonstrable conformational effect, and the structure reveals how occupancy of only one of the two feedback inhibition sites can immobilize the catalytic activity of both TrpE subunits. The presence of effectors in the structure provides a view of the locations of some of the amino acid residues in the active sites. Our findings are discussed in terms of the previously described AS structure of S. solfataricus, mutational data obtained from enteric bacteria, and the enzymes mechanism of action.

End product control of tryptophan biosynthesis in extracts and intact cells of the higher plant nicotina tabacum var. Wisconsin 38

Abstract A study has been made of the control by end-product inhibition of the tryptophan biosynthetic pathway in cultured cells of the higher plant, Nicotiana tabacum , var. Wisconsin 38. Crude extracts of callus tissue obtained from N. tabacum catalyzed the conversion of chorismic acid to anthranilic acid, the first step unique to l -tryptophan version of chorismic acid to anthranilic acid, the first step unique to l -tryptophan biosynthesis. The catalytic activity was destroyed by heat and no reaction occured with the ommission of either glutamine of Mg 2+ . In cell extracts this catalytic activity was inhibited by l -tryptophan. The inhibition was competitive with chorismate. In experiments with intact cells the presence of high l -tryptophan pools reduced the flow of radioactive carbon from the precursor, shikimic acid, into biosynthetic L -tryptophan. The data obtained were consistent with the operation of end-product inhibition in vivo

Tryptophan synthase from Nicotiana tabacum

Abstract A study has been made of the properties of a tryptophan synthase ( l -serine hydro-lyase (adding indole), EC 4.2.1.20) obtained from cultured cells of Nicotiana tabacum var. Wisconsin 38. The data presented show that the enzyme more closely resembles the two-component bacterial type of tryptophan synthase in contrast to the single-component fungal enzyme. 1. 1. Km values for the substrates indoleglycerol phosphate, indole, l -serine, and pyridoxal phosphate are 0.11 mM, 0.016 mM, 34.0 mM, and 1.3 μM, respectively. The pH optimum for Reaction 2 activity ( indole + l -serine → l -tryptophan ) of the enzyme is 8.2. 2. 2. The enzyme can be separated into two components by differential precipitation with (NH4)2SO4. Component B alone catalyzes the conversion of indole to tryptophan and this activity is not stimulated by the addition of Component A. The combination of Components A and B is necessary for the conversion of indoleglycerol phosphate to tryptophan. Component A is heat-labile with a sedimentation constant of 3.1 S. Component B is heat-stable with a sedimentation constant of 5.5 S. 3. 3. Mixed complementation experiments with the A and B subunits of Escherichia coli and N. tabacum tryptophan synthase, as well as antibody neutralization experiments, indicate that the B protein has undergone less change than the A protein throughout the course of evolution.

The crystal structure of anthranilate phosphoribosyltransferase from the enterobacterium Pectobacterium carotovorum

The structure of anthranilate phosphoribosyltransferase from the enterobacterium Pectobacterium carotovorum has been solved at 2.4 Å in complex with Mn2+‐pyrophosphate, and at 1.9 Å without ligands. The enzyme structure has a novel phosphoribosyltransferase (PRT) fold and displays close homology to the structures of pyrimidine nucleoside phosphorylases. The enzyme is a homodimer with a monomer of 345 residues. Each monomer consists of two subdomains, α and α/β, which form a cleft containing the active site. The nature of the active site is inferred from the trapped MnPPi complex and detailed knowledge of the active sites of nucleoside phosphorylases. With the anthranilate (An)PRT structure solved, the structures of all the enzymes required for tryptophan biosynthesis are now known.

A rapid spectrophotofluorometric assay for indoleglycerol phosphate synthase

Abstract A rapid and sensitive spectrophotofluorometric assay for indoleglycerol phosphate synthase is presented. The method is based upon the fluorescence of the reaction product indoleglycerol phosphate and has two advantages over previously reported assays. It is more sensitive and is useful for measuring enzyme activities in extracts containing materials that prohibit the use of other methods.

Genetic determination of the antigenic specificity of tryptophan synthetase

Rabbit antiserum was obtained against the purified enzyme, tryptophan synthetase, from the fungus Neurospora crassa . With quantitative serological methods it was possible to demonstrate the gene products of a large class of mutant strains (CRM − , td mutants) which cross-react with the non-neutralizing fraction of the antiserum. Differences were also uncovered in a second class of mutant strains (CRM + , td mutants) hitherto considered antigenically identical. The results have shown a relationship between the position of the lesion within the td locus and the nature of the cross-reacting protein present in the CRM~ extract.

Immunochemical comparisons of mutant and wild-type α-subunits of tryptophan synthetase

Abstract The effects of single and double amino acid substitutions on the tertiary structure of the α-subunit of tryptophan synthetase from Escherichia coli have been investigated by comparing the reactions of mutant and wild-type enzymes with β 2 -subunit and with antibodies directed against the wild-type α-subunit. About half the mutants exhibited reduced binding affinities β 2 -subunit. In micro-complement fixation assays, however, every mutant reacted with antisera exactly like wild-type. The results suggested that, when present, tertiary structure changes in the mutant proteins tested were limited, possibly to the portions of the surface comprising the active site and the β 2 -subunit binding site.

Crystallization and purification of the enzyme anthranilate phosphoribosyl transferase

Anthranilate phosphoribosyl transferase from the bacterium Hafnia alvei has been crystallized. This enzyme is one of a small number that constitute the biosynthetic pathway for tryptophan. Large cubic crystals were grown at 4 degrees C by dialyzing away the glycerol from a protein solution that included ammonium sulfate, polyethylene glycol and glycerol. The crystals were much more temperature stable and resistant to X-ray deterioration than a previous, similar crystal form that had included glycerol. The crystals belong to the space group I432, a = b = c = 189 A (1 A = 0.1 nm). The ratio of the monomer molecular weight, 37,000, to the volume of the unit cell suggests that there is one homodimer per asymmetric unit. The crystals diffracted to a resolution of 3.0 A at the Stanford Synchotron Radiation Laboratory X-ray source.

An immunochemical analysis of the dissociation of the β2 component of Escherichia coli tryptophan synthetase

Abstract Conversion of E. coli tryptophan synthetase β 2 subunit to an enzymatically inert apo-derivative is accompanied by a change in immunochemical reactivity. Restoration of the cofactor, pyridoxal-5′-phosphate, simultaneously restores the enzymatic and serological characteristics to normal. This change in antigenic structure is detectable by micro-complement fixation but not by macro-complement fixation or precipitin analysis. Evidence was obtained that: (1) with respect to the antisera used in the experiments, pyridoxal-5′-phosphate is neither an antigenic determinant nor part of a determinant; (2) the reversible shift in antigenic structure can best be explained by the enhanced dissociation of the apoenzyme to a strongly cross-reacting monomer; and (3) the tertiary structure of the monomer remains essentially unchanged upon dissociation.