Covalent Attachment of FAD Derivatives to a Fusion Protein Consisting of 6-Hydroxy-D-nicotine Oxidase and a Mitochondrial Presequence

Abstract

Autoflavinylation of 6-hydroxy-D-nicotine oxidase (6-HDNO) was successfully employed to modify the protein covalently with FAD derivatives. The model compounds N 6 -(2-aminoethyl)-FAD and N 6 -(6-carboxyhexyl)-FAD were spontaneously bound to a fusion protein consisting of the mitochondrial targeting sequence of Neurospora crassa F 0 -ATPase subunit 9 (Su9) attached to 6-HDNO. When translated in the rabbit reticulocyte lysate, Su9 -6-HDNO was in the trypsin-sensitive apoenzyme form; when translated in the presence of flavins it adopted a trypsin-resistant conformation characteristic of the 6-HDNO holoenzyme. With flavin derivatives, Su9 -6-HDNO exhibited approximately 50% of the 6-HDNO activity observed with FAD.The covalently modified Su9 -6-HDNO was imported into Saccharomyces cerevisiae mitochondria with an efficiency equal to that of the apoenzyme. Apparently the increase in size and charge of the FAD moiety did not hamper translocation across the mitochondrial membranes. Yeast mutant ssc1-2 mitochondria deficient in mtHsp70 unfoldase activity imported the flavinylated Su9 -6-HDNO protein. In mutant ssc1-3 mitochondria deficient in both mtHsp70 unfoldase and translocase activity Su9 -6-HDNO was trapped as translocation intermediate; the Su9 presequence was passed to the matrix where it was proteolytically cleaved by the mitochondrial processing peptidase; (MPP); the translocation-arrested 6-HDNO moiety adopted a trypsin-sensitive conformation. Our results indicate that unfolding of the FAD-stabilized flavin-binding domain of 6-HDNO in passage through the mitochondrial general insertion pore does not require the activity of mtHsp70.6-Hydroxy-D-nicotine oxidase (6-HDNO 1 ) belongs to a group of flavoenzymes with covalently attached FAD (for a review, see Ref. 1). Formation of the histidyl(N 3 )-(8␣)FAD bond proceeds autocatalytically and depends on a flavinylation-competent conformation of the protein (2, 3). The autocatalytic nature of the process opens the possibility to bind FAD derivatives to the active site His 71 of the enzyme in a spontaneous reaction. One aim of the present work was to test this assumption with in vitro synthesized apoenzyme, N 6 -(2-aminoethyl)-FAD, and N 6 -(6-carboxyhexyl)-FAD (4) and to establish how these substitutions affect folding and enzyme activity of the protein. A second aim was to use the protein-bound flavin derivatives as probes in an attempt to determine whether the increase in size and charge of the FAD moiety affects mitochondrial import of 6-HDNO supplied with a mitochondrial targeting sequence. Incorporation of FAD into the flavin-binding domain of 6-HDNO induces a conformational change in the trypsin-sensitive apoenzyme that renders the holoenzyme trypsin-resistant (5). 2 Based on this observation, we used 6-HDNO as a model flavoenzyme to approach a third aspect in this work, namely whether the FAD-stabilized trypsin-resistant conformation of holo-6-HDNO requires functionally active mtHsp70 for its unfolding during mitochondrial import. To address this question, we ...

Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: The current state of affairs

“…Recent studies have focused on the requirement for flavinylation in the mitochondrial import of 6HDNO-dimethylglycine dehydrogenase fusions (Stoltz et al, 1995), the interaction of 6HDNO with the chaperone protein GroE (Brandsch et al, 1992), and the covalent incorporation of flavin analogues (modified in the adenine moiety of FAD) into the active site of the enzyme (Stoltz et al, 1996a(Stoltz et al, , 1996b. In oxidase, where the FAD-linking His residue was replaced with Cys, noncovalently bound FAD could be displaced rapidly by added 8-methylsulfonyl-FAD or 8-chloro-FAD (Stolz et al, 1996b).…”

One-Step Immobilization of Glucose Oxidase in a Silica Matrix on a Pt Electrode by an Electrochemically Induced Sol−Gel Process

“…To fabricate high-performance enzyme-based biosensors, enzyme immobilization is a key step. The conventional enzyme-immobilization protocols involve physical adsorption, covalent attachment, and entrapment or encapsulation within a polymer membrane or inorganic matrixes . Each of these methods has its own advantages and limitations.…”

Characterization of a Highly Conserved FAD-binding Site in Human Monoamine Oxidase B

“…Some examples include succinate dehydrogenase (33), dimethylglycine dehydrogenase (34), 6-hydroxy-D-nicotine oxidase (35), trimethylamine dehydrogenase (36), p-cresol methylhydroxylase (37), and MAOs A and B (18). Some studies on covalent flavinylation of proteins indicate that covalent coupling of FAD to its respective proteins appears to be an autocatalytic reaction (18,(35)(36)(37)(38).…”