Invertase in a thermophilic fungus, thermomyces lanuginosus: its unusual behaviour And regulation

Abstract

The understanding of the phenomenon of thermophily requires investigations of both thermophilic prokaryotes and eukaryotes. In the eukaryotes, thermophily is exhibited only by a few species of fungi which can grow up to 60°C. A comparative study of homologous enzymes from thermophilic and mesophilic fungi and the analysis of the observed differences is a useful approach not only in discerning the mechanisms in thermophily but also in understanding the features of fungal growth and metabolism. Because of the availability of background information of invertase from some mesophilic sources and the convenience of assaying the enzyme, it was chosen for the projected study in a thermophilic fungus, Themomyces lanuginosus. The behaviour of invertase in the thermophilic fungus differed from invertases of mesophilic organisms in several respects, e.g., in the thermophilic fungus the enzyme was induced only in the presence of its substrate; it was intracellular and it was unstable both in mycelia and in cell-free extracts. The enzyme specific activity was maximum in 6 h-sucrose-grown mycelia following, which it progressively declined before maximal increase in biomass occurred and much of the inducer (sucrose) was still present in the growth medium. Further, invertase activity in cell-free extracts was unstable; it was completely inactivated during storage for 3 days at O°C. The enzyme activity was stabilised by the addition of thiol compounds, dithiothreitol (DTT) and glutathione (GSH) to cell-& extracts. In contrast, the addition of disulphides and thiol-modifying compounds rapidly inactivated the enzyme indicating the involvement of free sulphydryl group(s) in enzyme activity. The enzyme activity was reciprocally modulated by reduced (GSH) and oxidized (GSSG) glutathione, suggesting that invertase may be regulated by thiol/disulphide exchange reaction. Such a modulation of invert- activity has not been reported hr any other invertase. This observation suggested that the enzyme in the thermopbilic fungus is different from invert- that have been studied from mesophilic sources, notably from yeast and Neurospora. To obtain more information on this unusual behaviour of invertase of T. lanuginosus, an attempt was made to purify the enzyme and study its physico-chemical properties. Invertase was purified by ammonium sulphate fractionation of cellular proteins, ion-exchange and thiol-affinity chromatography followed by preparative electrophoresis. The final preparation of invertase after the electroelution step gave a single band on a native PAGE. However, the same preparation of invertase resolved into five bands of different molecular mass. The heterogeneity of the enzyme preparation on SDS-PAGE raised two possibilities with respect to the purity of the enzyme: (1) the final preparation contained multiple invertases of different molecular mass, or (2) the invertase preparation was associated with contaminating proteins. To distinguish between these two possibilities, proteins from induced (sucrose-grown) and non-induced (glucose-grown) mycelia were compared after identical steps of purification. The rationale of this experiment was that if heterogeneity of invertase is due to multiple forms of the same enzyme, they would most likely be absent in the non-induced mycelia. When the final preparations of proteins from both the mycelia were analysed on SDS-PAGE, it was observed that certain proteins were present in both the induced and the non-induced mycelia, suggesting that they might be the contaminating proteins present in the invertase purified by the above procedures. Some physico-chemical properties of invertase were studied. The purified enzyme was unstable during storage, losing activity completely in five days at O°C. Addition of DTT or glutathione did not prevent this loss of enzyme activity. This response of purified invertase preparations to DTT was quite opposite to that in cell-free extracts where invertase activity was stabilised by thiol compounds. To elucidate the reason for this difference in the behaviour of invertase in cell-free extracts and in pursed preparations, the approach taken was to first inactivate the enzyme in both1 type of preparations and then attempt to reactivate it. Dialysis of cell-free extracts had been found to cause an accelerated and complete inadivation oft he enzyme. The same treatment also inactivated freshly purified invertase, but to a lesser extent (60%). Whereas addition of DTT completely reatored the enzyme activity in the dialysed cell-bee extracts, it caused only a marginal revival of activity in dialysed invertase. This change in the response of purified invertase to DTT suggested that some cellular proteins were required br the reactivation of the enzyme by DTT that had been removed during the purification of invertase. A cellular protein was identified that reactivated inactive invertase in the presence of DTT. This protein was given the acronym "PRIA" for 'protein which restores i nvertase activity'. The mechanism of reactivation involved the conversion of the inactive invertase molecules into an active form. A model has been proposed to explain the requirement of UPRIA" for the reactivation of invertase. The salient features of this model are : (i) invert= requires free sulphydryl group(s) for activity, (ii) inactivation of invertase involves the formation of intramolecular disulphide bond(s) in the enzyme, (iii) the disulphide bond(s) is inaccessible to reduction by DTT, (iv) interaction of invertase and "PRIA” results in a conformational change in the enzyme that exposes the disulphide bond(s), rendering it susceptible to reduction by DTT and converting inactive invertase into active enzyme molecules. A surprising observation was the resistance of purified invertase to inactivation by the disulphides, GSSG, CoASSCoA and cystine. This was in marked contrast to their effective inhibition of invertase in the cell-& extracts. The experimental analysis of this unexpected resistance of purified invertase to disulphides revealed that following thioldnity chromatography on a Afegeldol column, invertase became resistant to disulphide inactivation. Moreover, the purified invertase was more stable during storage and to dialysis treatments in contrast to invertase activity in the cell-free extracts. These obsemtions suggested that invertase was altered- presumably it underwent a conformational change during the -el-501 chromatography step; possibly, the interaction of invertase with the gel matrix resulted in some cysteine residues in the enzyme becoming inaccessible to oxidation, thereby conferring resistance to inactivation by disulphides. The in vitro modulation of invertase activity by GSH and GSSG suggested the possibility that the enzyme may be regulated by a similar mechanism in the fungal mycelium. To substantiate this, GSH and GSSG levels in the mycelia were estimated. The GSH/GSSG ratio dememed in the mycelia between 6 and 18 h of growth and this was correlated with the decline in invertase activity. The fall in the GSHIGSSG ratio suggested that the intracellular environment waa becoming progressively oxidised during growth. Because NADPH participates in maintaining the cellular glutathione in a reduced state by the glutathione reduct- reaction, NADPH and NADPt levels were estimated. The NADPH/NADPt ratio declined by a factor of four between 6 and 36 h of growth and this decrease was positively correlated with the decrease in the flux of glucose through the pentose phosphate pathway. Incorporation of 'H-thymidine in mycelia indicated that with age of the culture, the number of growing hyphal tipslunit weight of mycelia declined. An attempt was made to integrate the changes in various biochemical parameters with the pattern of invertase development in T. lanuginosus when grown in a medium containing sucrose, i.e. invertase activity appeared rapidly as soon as perceptible growth occurred but it did not increase in parallel with the increase in biomass. Rather, the activity started to decline at approximately 6 h at which time growth was quantitatively mall. Since invertase activity in T. lanuginosus was induced by sucrose which is transported inside by a specific transporter, the development of invertase activity was linked to the uptake of sucrose by the fungal mycelia. It was considered likely that the sucrose transporter in T. lanuginosus, is localised at the hyphal tip where the entry of sucrose induces invertase. He enzyme is kept active in the hyphal tip because of a reductive environment due to a high GSHIGSSG ratio as a result of high NADPH levels. The latter serves to maintain GSH in a reduced state by the glutathione reductase reaction. In mature hyphae, lower generation of NADPH will result in lower GSHIGSSG ratio that will inactivate invertase by thiol oxidation. According to this model, the early burst of invertase activity in sucrose grown T. lanuginosus mycelia is due to the initiation of branch initials whereas the fall in enzyme activity is because of the decline in the proportion of hyphal tips per unit mass of mycelium as elongation growth and wall thickening occurs.