Proteolytic Cleavage of Hansenula anomala Flavocytochrome b₂ into Its Two Functional Domains

Abstract

In a previous work, we have described the tryptic cleavage of yeast flavocytochrome b, into its two functional domains: a cytochrome h, core and a flavodehydrogenase. The lactate dehydrogenase efficiency of the latter was, however, dramatically low, only about 1 :< that of intact flavocytochrome b,. Our present study concerns a new flavodehydrogenase derivative of Hansenula anomala flavocytochrome 6 , which spontaneously dissociates from the cytochrome domain when the polypeptide bridge connecting thein is cleaved by Staphylococcus aureus V 8 protease I. This flavodehydrogenase was purified and some of its functional and structural properties were studied. It presents an exceptionally high lactate dehydrogenase activity, about 80 "/, that of flavocytochrome b,. This result clearly demonstrates that the cytochrome domain is not necessary for the lactate dehydrogenase function and suggests an autonomous folding for both domains.Our results are discussed in terms of 'gene fusion'.Each chain of yeast flavocytochrome b, has been shown to be folded into three globules: n, E and / I ' joined together in the 'intact' enzyme by two protease-sensitive bridges aa' and cd [I -41 (cf. These two functional domains, flavodehydrogenase and cytochrome, which spontaneously dissociate from each other when their connecting bridge cd is cleaved by trypsin, were isolated as stable, pure, molecular entities, termed FDH, and nT respectively [2, 3, 91. Unfortunately, this polypeptide bridge cd is not the part of the native chain which is most sensitive towards trypsin: the aa' zone is cleaved about 20-times faster [lo], so that FDHT was always found altered by this undesirable inner cleavage, which was achieved with a loss of about 20 amino acid residues [3].From a functional point of view, the first cleavage in aa' results in a 96 %loss of activity [I I]; the isolated 'nicked' FDH, entity itself only retains about 1 % of the ferricyanide reductase activity of native flavocytochrome b2 [lo]. These results did not elucidate whether such a low activity was due to the aa' cleavdge itself or to the release of cytochrome b2 core resulting from the second cleavage at cd.This study presents information concerning this question. Despite the hypersensitivity of the aa' zone towards a number of proteases [I2 -141, we succeeded in obtaining a cleavage in the cd zone faster than in the aa' zone. This was achieved with Hansenula anomala flavocytochrome b2 and the highly specific Staphylococcus aureus V 8 protease 1. We describe a method for separating the so-formed 'intact' flavodehydrogenase part which we currently term 'FDH,,', from the other proteolysis products. Steady-state catalytic parameters of this new flavocytochrome h, derivative have been determined and compared with those of the native flavocytochrome b, and its tryptic derivatives. MATERIALS A N D METHODS Hansenula anomala Flavocytochrome b,This was prepared according to [I51 with the minor modifications described in [I I]. It was stored as an ammonium sulfate precipitate (50 % satur...

Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b2 after mild proteolysis by an H. anomala proteinase

“…Its reaches 65% of the expected amount after a 7-h incubation. By SDSjPAGE it was assigned to the n fragment, suggesting that F1 is the flavoprotein domain and that the cleavage giving rise to F1 and n occurred in the cd zone according to our present knowledge of flavocytochrome h2 structure [12]. Although not demonstrated in this particular case, the n' fragment might be a shorter form of the cytochrome core as was previously observed in the case of tryptic digestion [16].…”

“…The yield of this proteolytically obtained flavoprotein domain relative to the initial amount of flavocytochrome b2 is usually higher than 20%, which is one order of magnitude more than that obtained with S . aweus V8 protease I [12].…”

“…Unfortunately, it has been difficult to obtain a highly active FDH domain because of the existence of another proteolytically sensitive zone (aa', see Fig. 1) 1141, the cleavage of which results in a dramatic decrease in activity [14-161. This difficulty can be partly overcome by using Staphylococcus aweus V8 protease I which cleaves primarily the cd zone [12]. However, the procedure developed did not yield large amounts of the flavodehydrogenase moiety.…”

Study of the Hansenula anomala yeast flavocytochrome‐b2‐cytochrome‐c complex

“…cm-' [15]. The FDH,, flavodehydrogenase concentration was determined from tryptophan fluorescence in 6 M guanidine as described [13]. Each fragment sample was desalted prior to use by dialysis (overnight) against 10mM phosphate buffer at 4°C with or without 10 mM sodium 111.-lactate, pH 7.2.…”

“…Buffers Fla~~orlehydrogeiia.sc. The flavodehydrogenase fragment (FDH,,) was obtained by chromatography after proleolytic cleavage of flavocytochrome h, by the S. aureus V8 protease I (100 pg/ml protease, 100 pM flavocytochrome h,, 75 mM ammonium acetate, 100 pM lactate buffer, pH 7.8) for 140 min at 20 'CfollowingtheprocedureofGervaiset al [13]. The FDH,, fragment was separated from the cytochrome b, core (nv8) and from uncleaved chains by gel filtration through Sephadex G-100 followed by ion-exchange chromatography on DEAEcellulose in phosphate/urea dissociating buffer.…”