Crystal Structure of the Plant PPC Decarboxylase AtHAL3a Complexed with an Ene-thiol Reaction Intermediate

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Abstract

The Arabidopsis thaliana protein AtHAL3a decarboxylates 4′-phosphopantothenoylcysteine to 4′-phosphopantetheine, a step in coenzyme A biosynthesis. Surprisingly, this decarboxylation reaction is carried out as an FMN-dependent redox reaction. In the first half-reaction, the side-chain of the cysteine residue of 4′-phosphopantothenoylcysteine is oxidised and the thioaldehyde intermediate decarboxylates spontaneously to the 4′-phosphopantothenoyl-aminoethenethiol intermediate. In the second half-reaction this compound is reduced to 4′-phosphopantetheine and the FMNH2 cofactor is re-oxidised. The active site mutant C175S is unable to perform this reductive half-reaction. Here, we present the crystal structure of the AtHAL3a mutant C175S in complex with the reaction intermediate pantothenoyl-aminoethenethiol and FMNH2. The geometry of binding suggests that reduction of the CαCβ double bond of the intermediate can be performed by direct hydride-transfer from N5 of FMNH2 to Cβ of the aminoethenethiol-moiety supported by a protonation of Cα by Cys175. The binding mode of the substrate is very similar to that previously observed for a pentapeptide to the homologous enzyme EpiD that introduces the aminoethenethiol-moiety as final reaction product at the C terminus of peptidyl-cysteine residues. This finding further supports our view that these homologous enzymes form a protein family of homo-oligomeric flavin-containing cysteine decarboxylases, which we have termed HFCD family.

Introduction

The release of carbon dioxide from carbonic acids by decarboxylases is a frequently occurring reaction in biochemistry. This is reflected by 78 entries in the ENZYME data base for this enzyme class (EC 4.1.1.-).1 Many representatives depend on cofactors including thiamine pyrophosphate (TPP), biotin, pyruvate or pyridoxal phosphate (PLP).

The decarboxylation of 4′-phosphopantothenoylcysteine (PPC) to 4′-phosphopantetheine (PP) is a key step in coenzyme A biosynthesis from pantothenate.2 For Escherichia coli, rat and horse this decarboxylase activity was first assigned to pyruvoyl-dependent enzymes.3., 4., 5., 6. However, no gene encoding a pyruvoyl-dependent PPC decarboxylase could be identified and it was not clear how the pyruvoyl group could contribute to this reaction as there is no free amino group in the substrate. None of the proposed reaction mechanisms was verified.7., 8.

Finally, two years ago it could be demonstrated that the PPC decarboxylases (EC 4.1.1.36)1 are not pyruvoyl but flavin-dependent enzymes. For E. coli, it was shown, that the NH2-terminal domain of the Dfp flavoprotein (the CoaC domain) catalyses the decarboxylation of PPC.9 Dfp is a bifucntional enzyme and also has PPC synthetase activity that resides in the COOH-terminal CoaB domain.10., 11. Later it was confirmed that flavin-dependent decarboxylation of PPC also occurs in plant12 and human coenzyme A biosynthesis.13 The plant PPC decarboxylase AtHAL3a was originally identified as a flavoprotein related to plant growth and salt and osmotic tolerance.14

Plant and human PPC decarboxylases share the proposed PPC decarboxylase signature pattern with the CoaC domain of the Dfp proteins.15 However, there is also sequence homology with the LanD flavoproteins EpiD from Staphylococcus epidermidis and MrsD from Bacillus.9 Both enzymes catalyse the oxidative decarboxylation of peptidylcysteine residues to peptidyl-β-aminoethenethiol,16., 17. a reaction involved in biosynthesis of the lantibiotics epidermin and mersacidin. Lantibiotics are ribosomally synthesised and posttranslationally modified antibiotic peptides.18 The PPC decarboxylases and the LanD enzymes belong to a new family of flavoproteins which was named HFCD (homo-oligomeric flavin-containing Cys decarboxylases).9., 19. HFCD proteins share conserved active-site residues, a new flavin-binding mode and are trimeric (AtHAL3a) or dodecameric proteins (EpiD, MrsD and Dfp). All members of this protein family characterised so far catalyse the decarboxylation of cysteine residues.

The central point in characterising the HFCD proteins is to elucidate the exact role of the flavin cofactor for the decarboxylation mechanism. We had already proposed that the flavin cofactor oxidises the side-chain of the cysteine residue16 and that decarboxylation of cysteine residues occurs spontaneously via the thioaldehyde tautomer of the oxidised side-chain.9 The crystal structure of the active-site mutant EpiD H67N with a bound substrate peptide then revealed the surprising result that Cα–Cβ dehydrogenation by FMN and subsequent tautomerisation does not take place. In contrast, the geometry of the enzyme–substrate complex suggested that the oxidative attack of FMN starts at the thiol, yielding directly the thioaldehyde intermediate.19

The detection of oxidatively decarboxylated reaction products for the homologous peptidylcysteine decarboxylases EpiD and MrsD was a direct hint that PPC decarboxylases also follow the mechanism “decarboxylation by initial oxidation”, although the final product PP is not oxidised. Based on the crystal structure of EpiD H67N with bound substrate peptide19 and the crystal structure of AtHAL3a,20 a model for binding of PPC to AtHAL3a has been proposed.12 In this model binding of PPC involves a binding clamp that is disordered in the absence of substrate. Using the unphosphorylated substrate pantothenoylcysteine (PC) and AtHAL3a proteins, in which residues of this substrate binding clamp were exchanged, it was possible to identify the proposed oxidatively decarboxylated intermediate by high-resolution mass spectrometry and UV–visible spectroscopy.21 It is reasonable to assume, that the decarboxylation mechanism is not dependent of the used substrate (PC or PPC), although the oxidatively decarboxylated intermediate was not present in assignable amounts for PPC. We also suggested that Cys175 of AtHAL3a, which is conserved in the PPC decarboxylases but not the LanD enzymes, is responsible for reduction of the oxidatively decarboxylated intermediate and/or re-oxidation of the flavin cofactor yielding the final product.21

The three-dimensional structures of AtHAl3a20 and EpiD19 have been determined but only the latter structure revealed the substrate binding mode and the reaction mechanism of LanD proteins acting on peptide substrates. To get insights into the binding mode of substrate and the reaction mechanism of PPC decarboxylases, we analysed crystals of the mutant AtHAL3a C175S soaked with PC. PC was oxidatively decarboxylated by this enzyme and due to the mutation of the catalytic Cys175 residue, re-oxidation of the simultaneously reduced cofactor FMN was not observed. Here, we present the high-resolution structure of AtHAL3a-C175S-FMNH2 with bound oxidatively decarboxylated PC (pantothenoylaminoethenethiol). Based on this crystal structure we propose a mechanism for re-oxidation of FMNH2 and reduction of the oxidised intermediate. The data presented here, together with recently published data9., 16., 17., 21., 22. confirm that the decarboxylation of cysteine residues in PPC decarboxylases is a flavin-dependent reaction, although there is no overall redox reaction.

Section snippets

Complex formation

The mutant AtHAL3a C175S has been demonstrated to perform only one half-reaction, the oxidative decarboxylation of PC, but is unable to complete the reaction to form the decarboxylated product pantetheine.21 During this half-reaction FMN is reduced to FMNH2 (Figure 1). This property opened the possibility to analyse AtHAL3a in complex with this reaction intermediate, which has the ene-thiol group in common with the end product of LanD proteins like EpiD.16 As AtHAL3a decarboxylates both, PC and

Conclusion

We could demonstrate that two subgroups of the HFCD family, the LanD proteins including EpiD, and the PPC decarboxylases including AtHAL3a, share general principles of substrate recognition and conversion. The substrates have a terminal cysteine residue in common and are embraced in an almost extended conformation by a substrate recognition clamp that is disordered in the absence of ligands. The major difference in the reaction products arises from the ability of the PPC decarboxylases to

Materials and Methods

The AtHAL3a mutant C175S was expressed in E. coli and the recombinant protein was purified21 and crystallised20 essentially as described earlier but without reducing agents. Obviously C175, which is located in a flexible loop structure, is responsible for the formation of higher aggregates of the wild-type under oxidising conditions. These could be easily detected by analytical gel permeation chromatography, whereas the C175S mutant was monodisperse. The purified protein was of greenish colour.

Acknowledgements

We thank Michael Uebele for synthesis of D-pantothenoylcysteine and Jens T. Kaiser for critical reading of the manuscript. This work was supported by Deutsche Forschungsgemeinschaft SFB 413 to St. S. and Grant KU869/6-1 to T.K., by a fellowship from the European Molecular Biology Organization to P.H.-A. (ASTF 9878), by EU QLK3-CT-2001-01976 to R.H. and Spanish MCT Grant BMC2002-03128 to F.A.C.-M.

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Present address: S. Steinbacher, Division of Chemistry and Chemical Engineering, Mail Code 114-96, California Institute of Technology, Pasadena, CA 91125, USA.

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