Frank H. Gaertner

A cluster-gene: Evidence for one gene, one polypeptide, five enzymes☆

Abstract Five enzymes catalyzing consecutive steps in the central pathway leading to the biosynthesis of the aromatic amino acids in Neurospora are physically associated. Previous structural analysis of this enzyme system suggested that the 300,000 dalton native protein consisted of at least four distinct polypeptide chains. Genetic studies conducted in another laboratory indicated that the genes specifying these putative polypeptides are tightly linked. Recently we found that the “subunit” structure observed earlier for this system was created during its isolation and purification by the action of resident proteolytic activity. Here we show that when the enzyme system is purified rapidly by affinity chromatography in the presence of protease inhibitor a single 150,000 dalton band is obtained in dodecyl sulfate gel electrophoresis. Without these precautions, four or more distinct bands with lower molecular weights are obtained. From these results we conclude that the arom enzyme system is a 300,000 dalton dimer consisting of identical 150,000 dalton peptides. Based on the one gene, one polypeptide concept, the arom system appears to represent a case of one gene specifying five enzyme activities rather than five separate genes as previously conceived. The term cluster-gene is proposed to signify a gene encoding a polypeptide with discrete multiple functions.

Chorismate synthase of Neurospora crassa: A flavoprotein

Abstract Chorismate synthase is purified from Neurospora crassa. The final step is accomplished by preparative electrophoresis. Its purity is estimated at ≥90% on the basis of analytical polyacrylamide gel electrophoresis. The enzyme appears to be active in at least two multimeric states, with a subunit molecular weight of ~55,000. The purified enzyme preparation is absolutely dependent on the presence of a reducing system, which can readily be provided under aerobic conditions by NADPH plus FMN or under stringent anaerobic conditions by dithionite. The following evidence implicates a physiological role for FMN in N. crassa chorismate synthase activity: (a) a preferential stimulation of activity by NADPH and FMN over other pyridine and flavin nucleotides, respectively, in both impure and purified enzyme preparations; (b) an alteration of the Chromatographic pattern of the enzyme on diethylaminoethylcellulose by the addition of FMN to the elution buffer; (c) an apparent binding of FMN to the enzyme as exhibited by gel filtration in the presence of the substrate, 3-enolpyruvylshikimate 5-phosphate; (d) a requirement for preliminary incubation with FMN, in concert with the substrate, to eliminate a reaction lag (i.e., to activate the enzyme); (e) a substrate-dependent diaphorase activity exhibited by purified enzyme preparations in the presence of FMN and NADPH. The observed activation and alteration of Chromatographic behavior of chorismate synthase by FMN suggest that the flavin nucleotide influences the conformation of the enzyme. The ability to replace NADPH and FMN with dithionite suggests that FMN mediates the flow of electrons from a source of reducing power (NADPH) to some enzymic site important to the function of the enzyme. Hence, the diaphorase activity which is observed as intrinsic to chorismate synthase of N. crassa may be significant from the standpoint of catalysis or may have importance as a regulatory mechanism.

The protease problem in Neurospora: Structural modification of the arom multienzyme system during its extraction and isolation☆

Abstract The “aromatic complex” or “ arom aggregate” of Neurospora crassa catalyzes five consecutive reactions in the central pathway leading to the biosynthesis of the aromatic amino acids. Previously, this multienzyme system was shown variously to have a molecular weight of 230,000 to 300,000 and to contain up to four subunits. Recently, a protease and a corresponding specific inhibitor have been isolated from N. crassa and, as described in this report, a new method for isolating the multienzyme system has been developed. We have made the following observations: (a) Detergent (sodium dodecyl sulfate) gel electrophorograms of the “complex” isolated by two different methods are not comparable. In an earlier method, which involved more manipulations and time, the detergent gel banding patterns showed four polypeptides with molecular weights totaling about 300,000. With the new purification procedure, there are two major bands: the first with an apparent molecular weight of about 150,000 and the second with a molecular weight of 50,000. (b) When the freshly purified multienzyme system is incubated at 25 °C, four new bands appear within 30 h and a fifth is visible after 40 h. (c) The formation of these new bands is prevented for up to 40 h by the addition of phenylmethanesulfonylfluoride or a purified preparation of the specific N. crassa protease inhibitor, (d) The multienzyme system appears to remain intact, as shown by standard polyacrylamide gel electrophoresis, even after it has suffered several proteolytic clips. These results demonstrate that the purified complex is contaminated with a small but influential quantity of the inhibitable N. crassa protease and show that this protease is capable of creating an artificial subunit structure in the multienzyme system. Based on these observations, we hypothesize that the arom enzyme system is a five-component multifunctional enzyme.

Enzyme Organization in the Polyaromatic—Biosynthetic Pathway: The arom Conjugate and Other Multienzyme Systems*

Publisher Summary The multienzyme system involved in the de novo biosynthesis of aromatic amino acids, particularly as it exists in many eukaryotic microorganisms, truly represents a “model system” for studying the structure and function of enzyme organization in vitro . The synthesis of tryptophan requires a total of 13 enzyme reactions, seven of which are constituted by the polyaromatic branch. In Neurospora , for example, this pathway yields three isolable multienzyme clusters—one of which is the arom conjugate. Each of the three clusters exhibits unique kinetic and/or regulatory properties, such as, metabolite “channeling” and coordinate activation. Studies on organized enzyme systems can be expected to have a major impact in the area of metabolic regulation, as well as in the areas of genetics and molecular evolution. This chapter discusses some of the important regulatory features of enzyme organization.

Purification of two multienzyme complexes in the aromatic/tryptophan pathway of Neurospora crassa

Abstract A procedure originally designed to isolate the anthranilate synthetase complex of Neurospora crassa is shown to yield also the aromatic complex in homogeneous form. The ionic and physical properties of the two complexes are shown to be similar. Through the use of parallel sedimentation-equilibrium analyses in H 2 O and D 2 18 O, the aromatic complex is shown to have a partial specific volume of ~0.72 and a molecular weight of ~290,000. Disk-gel electrophoresis in sodium dodecyl sulfate appears to show that the complex dissociates in the detergent into four unequal components with molecular weights totaling ~290,000. Apparent differences in physical properties were found between the aromatic complex isolated here and the aromatic complex isolated from N. crassa by other investigators.

Phosphocellulose, an affinity chromatographic system for chorismate synthase and the aromatic complex of Neurospora crassa☆

Abstract Unlike other enzymes of the aromatic multienzyme system, chorismate synthase and the aromatic complex of Neurospora crassa were found to bind to a column of cellulose phosphate and to elute at a relatively high concentration of phosphate (∼ 0.2 M ). The fact that other enzymes with similar ionic properties failed to bind to phosphocellulose suggests that the binding of the former two enzyme systems is due to a specific affinity for phosphate. This conclusion is not only supported by the fact that these same enzymes did not bind to a column of carboxymethyl cellulose, but also is consistent with the nature of the catalytic reactions of the enzymes. Both the shikimate kinase enzyme of the aromatic complex and chorismate synthase would be expected to have active sites which accomodate a phosphate moiety. We anticipate that other enzymes which involve phospho-substrates will also be amendable to this procedure.

Proteolytic inactivation of a pentafunctional enzyme conjugate: Coordinate protection by the first substrate

Abstract The arom pentafunctional enzyme conjugate of Neurospora crassa was exposed to trypsin, chymotrypsin, or a protease preparation from Neurospora in the presence and absence of the first substrate, 3-deoxy-D- arabino -heptulosonate 7-phosphate. It was found that the first substrate coordinately protects all five activities from proteolytic inactivation, which indicates a conformational change induced by this compound. In addition, the data presented are consistent with the “domain” theory of conjugate structure. It is also argued that coordinate protection may be of physiological significance.

Sedimentation properties of anthranilate synthetase from osmotic lysates of a wall-less variant of Neurospora

Abstract Anthranilate synthetase from osmotic lysates of a wall-less variant of Neurospora crassa is shown to have a sedimentation coefficient larger than 10.35 — the value obtained for the same enzyme isolated from wild-type strains by relatively harsh extraction procedures. Sucrose gradients performed in a nitrogen atmosphere indicate that the lysates contain anthranilate synthetase which sediments up to 15S. It is implied that the more gentle extraction procedure used with the wall-less variant maintains a greater organizational integrity of the enzyme.

A Cryobiological Method for the Enrichment of Fungal Mutants

In a previous abstract we reported the successful application of cryobiological techniques to the recovery of auxotrophic mutants of Neurospora crassa from a mixture of wild-type and mutant conidia (Leef & Gaertner, 1975). This communication presents in greater detail the methods used and the pertinent physiological observations which led to the development of the technique. We also show that by using cryobiological techniques it is possible to enrich for auxotrophs by means of the effects of either dehydration or the formation of intracellular ice.