Enzymatic Alkaloid Diversification



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Enzymatic Alkaloid Diversification

DOI: 10.1002/anie.200((will be filled in by the editorial staff))

Structural Basis and Enzymatic Mechanism of the Biosynthesis of C9- from C10- Monoterpenoid Indole Alkaloids**

Liuqing Yang, Marco Hill, Meitian Wang, Santosh Panjikar, and Joachim Stöckigt*

Dedicated to Professor Heinz G. Floss on the occasion of his 75th birthday.

Plants are an extremely important source of alkaloids, with over 20,000 identified to date and classified into a number of distinct families.[1] Thanks to their long known therapeutic importance,[2,3] structural diversity[4] and complex biosynthesis,[4,5] the monoterpenoid indole alkaloid family attracted significant interest. Many alkaloids of the ajmalan and sarpagan subfamilies exhibit a C9- instead of a C10-monoterpenoid unit. Ajmalan type alkaloids, such as ajmaline, are important therapeutics to treat antiarrhythmic heart disorders. Since, like all monoterpenoid indole alkaloids, the members of these subfamilies are derived from the 3α(S)-glucoalkaloid strictosidine (1),[6,7] which displays the C10- secologanin skeleton, loss of a C1 unit during their biosynthesis must take place somewhere downstream of 1. We report here the determination of the X-ray crystal structure of polyneuridine aldehyde esterase (PNAE)[8,9] which reveals important mechanistic insight into the biosynthesis of both subfamilies.

I


[] L. Yang, Prof. Dr. J. Stöckigt
Institute of Materia Medica, College of Pharmaceutical Sciences, Zhejiang University, 383 Yu Hang Tang Road, Hangzhou 310058, P.R. China
Fax: +49 6131 3923752
E-mail: stoeckig@uni-mainz.de
Homepage: http://www.pharmazie.uni-mainz.de/AK-Stoe/index.html

L. Yang, Dr. M. Hill, Prof. Dr. J. Stöckigt


Lehrstuhl für Pharmazeutische Biologie, Institut für Pharmazie, Johannes-Gutenberg Universität, Staudinger Weg 5, D-55128 Mainz, Germany

Dr. M. Wang


Swiss Light Source PX Ⅲ, Paul Scherrer Institute, CH-5232 Villigen, Switzerland

Dr. S. Panjikar


European Molecular Biology Laboratory Hamburg, Outstation Deutsches Elektronen-Synchrotron, Notkestrasse 85, D-22603 Hamburg, Germany

[] This work is supported by Deutsche Forschungsgemeinschaft (Bad Godesberg, Germany), Fonds der Chemischen Industrie (Frankfurt/Main, Germany) and Zhejiang University K.P.Chao’s High-Tech Foundation (Hangzhou, PR China). We thank Prof. Dr. David E. Cane (Brown University, USA) for critical discussion.

Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author.
n order to carry out the biochemical characterization of PNAE, the enzyme was isolated from plant cell cultures of the Indian medicinal plant Rauvolfia serpentina,[10] partially sequenced, the PNAE cDNA cloned and overexpressed in Escherichia coli, as previously described.[8,9] PNAE showed extremely high specificity for its natural substrate, polyneuridine aldehyde (2), with only 2 being processed out of 13 alkaloidal and aromatic methylesters.[9] Sequence analysis allowed preliminary classification of PNAE as a new candidate of the large α/ß hydrolase superfamily.[9],[11] This classification is based in particular on the catalytic triad S87, D216, H244 which was previously verified by single mutations and homology modeling.[9] Together with several well known recently characterized enzymes,[12,13] the role of PNAE in alkaloid biosynthesis is shown in Scheme 1.


Scheme 1. Central role of polyneuridine aldehyde esterase (PNAE) in the biosynthesis of the C9-monoterpenoid ajmalan and sarpagan alkaloid subfamilies (biogenetic numbering, STR1 - strictosidine synthase, SG - strictosidine glucosidase, SBE - sarpagan bridge enzyme).

Mechanistically, the key chemical reaction of the enzyme is the hydrolysis of the methylester function of 2, leading to the postulated polyneuridine ß-aldehydoacid (2a). ß-Ketoacids, such as oxalosuccinate in the Krebs cycle, are highly unstable due to facile enzyme- or buffer-catalyzed decarboxylation, occur frequently as intermediates in biosynthetic pathways. Decarboxylation of 2a results in generation of the first C9-monoterpenoid alkaloid 16-epi-vellosimine (3), the direct biosynthetic precursor of the ajmalan subfamily in Rauvolfia. Epimerization of 3 results in vellosimine (4), which functions as an initial precursor in C9-sarpagan alkaloid biosynthesis. It is the loss of the C10-terpenoid skeleton carbon dioxide (C-22) occurring as a result of PNAE action that leads to the biosynthesis of C9-Rauvolfia alkaloids from the C10-progenitors 2 and 2a (Scheme 1).

Biochemical data provided evidence for the enzyme-catalyzed formation of both alkaloid groups, but direct insight into the 3D-structure and the mechanism of PNAE we are describing here.[13]

The overall crystal structure (Figure 1) shows the core domain of PNAE, which consists of six ß-sheets flanked by five α-helices. Together with the cap domain,[14] the 3D-structure unequivocally confirms for the first time that PNAE belongs to the well known α/ß hydrolase fold, but has a novel function. The opening of the reaction channel is located in the cap (supp. information), a region which in contrast to the canonical core of the α/ß hydrolases, is structurally highly flexible. The deep channel leads 2 to the catalytic active site residues which are in typical α/ß-fold order nucleophilic (S87) -acidic (D216) -basic (H244), suggesting a serine esterase mechanism (Scheme 2).






Figure 1. a. PNAE’s overall 3D-structure (PDB code 2WFL). b. Enzyme product 3 (cyan) covalently linked as semi-acetal to S87 in the active site of PNAE mutant H244A (PDB code 3GZJ), overlaid with H244, water molecule of native structure (yellow), of complex structure (red). Estergroup (yellow) is modeled. Cap domain illustrated in orange.

The PNAE H244A mutant shows extremely reduced hydrolase activity (~1.3% rel. activity compared to the wild type enzyme). As now demonstrated by the crystal structure of H244A in complex with the enzyme product 3, the indolic part in the molecule interacts with M113, F125, Y128 and L187. This arrangement enables the alkaloid to be fixed by hydrophobic, sandwich-like interactions providing optimal structural accommodation for catalysis. The optimized geometry of the active center, the substrate and C-17 aldehyde group are crucial for PNAE activity, since slight changes in structure or functionality result in loss of enzymatic hydrolysis, for example if 2 is compared with the indole alkaloid picralinal or with polyneuridine (reduced C-17 CHO) (supp. information).

In addition to the necessity of the catalytic triad as proven by site-directed mutagenesis,[9] residue M245 located in the binding pocket of PNAE is also indispensable for hydrolysis, since M245A mutant shows only ~0.4% rel. activity. M245 is far (~5Å) from the water molecules in the active center and as the neighbour residue of the catalytic H244, it might therefore be of structural rather than of catalytic significance. This situation seems to be similar to H86, the neighbour of S87, since mutant H86A also exhibits only ~0.1% rel. activity. After S87-assisted hydrolysis, the ß-aldehydoacid 2a (Scheme 2) decarboxylates to the enolized enzyme product 3, in which the enolate anion is stabilized by hydrogen bonds of the backbone amides of G19 and F88 forming an oxyanion hole. Of the three reasonable mechanisms for ß-ketoacid decarboxylation[15]: (a) metal ion catalyzed, (b) Schiff base-assisted and (c) hydrogen bond and /or electrostatic-based polarization of the ketogroup, (a) and (b) can be excluded. Divalent metal cation-complexing EDTA[10a] or borohydride reduction in presence of 2 which would reduce an intermediate Schiff base and block the catalysis, did not affect PNAE activity. Moreover, 3D structure of the enzyme and complex do not display Schiff base forming residues and the complex no metal ions ( no significant peak in anomalous Fourier map) in the active center, both results favouring also mechanism (c).

The structural data now available for PNAE support an overall mechanism as proposed in Scheme 2, which undoubtedly represents the key reaction for the biosynthesis of C9-monoterpenoid Rauvolfia alkaloids.

The data will also allow a rational, structure-based redesign of PNAE, similar as we have recently demonstrated for the Pictet-Spenglerase, strictosidine synthase,[16] which is now applied for chemo-enzymatic synthesis of novel alkaloid libraries (unpublished).

Because it is the cap domain (supp. information) and the architecture of the binding pocket of PNAE determining its exceptional high substrate specificity, future systematic structure-based, second-sphere and random mutations should give PNAE mutants with altered, especially low, substrate specificity. Such enzymes then could be useful multi-purpose catalysts for generation of novel alkaloid structures for biological screening.






Scheme 2. Proposed serine esterase-typical reaction mechanism catalyzed by PNAE is the basis for the biosynthesis of C9- from C10-monoterpenoid indole alkaloids of ajmalan and sarpagan subfamilies in the Indian medicinal plant Rauvolfia serpentina (partial structures of substrate 2 and product 3 are shown; the covalently linked enzyme product 3 is marked *; Average distances of residues are in Å).

Experimental Section

Experimental procedures, crystallographic data, 3D-structure and chemical structures are provided in the Supporting Information.

Received: ((will be filled in by the editorial staff))
Published online on ((will be filled in by the editorial staff))

Keywords: enzyme catalysis · indole alkaloid biosynthesis · polyneuridine aldehyde esterase (PNAE) · protein structures · reaction mechanism

[1] J. Buckingham, (Eds.) Dictionary of Natural Products on DVD, version 16.2,Chapman and Hall/CRC, Boza Raton, 2008.

[2] a) M. H. Zenk, M. Juenger, Phytochemistry. 2007, 68, 2757-2772; b) J. Leonard, Nat. Prod. Rep. 1999, 16, 319-338.

[3] E. J. Saxton, Nat. Prod. Rep. 1997, 14, 559-590.

[4] G. A. Cordell, The Alkaloids: Chemistry and Biology, Academic Press, San Diego, 1998, 50, p.260.

[5] S. E. O’Connor, J. Maresh, Nat. Prod. Rep. 2006, 23, 532-547.

[6] T. M. Kutchan, Phytochemistry, 1993, 32, 493–506.

[7] J. Stoeckigt, M. Ruppert in Comprehensive Natural Products Chemistry:Amino Acids, Peptides, Porphyrins and Alkaloids. Vol. 4 (Eds.: D. H. R.Barton, K. Nakanishi, O. Meth-Cohn, J. W. Kelly), Elsevier, Amsterdam, 1999, pp.109–138.

[8] E. Dogru, H. Warzecha, F. Seibel, S. Haebel, F. Lottspeich, J. Stoeckigt, Eur. J. Biochem. 2000, 267, 1397-1406.

[9] E. Mattern-Dogru, X. Ma, J. Hartmann, H. Decker, J. Stoeckigt, Eur. J. Biochem. 2002, 269, 2889-2896.

[10] a)A. Pfitzner, J. Stoeckigt, Planta Med. 1983, 48, 221-227; b)A. Pfitzner, J. Stoeckigt, Chem. Soc. Chem. Commun. 1983, 459-460.

[11] X. Cousin, T. Hotelier, K. Giles, J. P. Toutant, A. Chatonnet, Nucleic Acids Res. 1998, 26, 226-228.

[12] M. Ruppert, X. Ma, J. Stoeckigt, Curr. Org. Chem. 2005, 9, 1431-1444.

[13] a)J. Stoeckigt, S. Panjikar, Nat. Prod. Rep. 2007, 24, 1382-1400; b)J. Stoeckigt, S. Panjikar, M. Ruppert, L. Barleben, X. Ma, E. Loris, M. Hill, Phytochem. Rev. 2007, 6, 15-34.

[14] Y. Cajal, A. Svendsen, J. deBolos, S. A. Patkar, M. A. Alsina, Biochimie. 2000, 82, 1053-1061.

[15] a)G. J. Poelarends, W. H. Johnson, A.G. Murzin, C. P. Whitman, J. Biol. Chem. 2003, 278, 48674-48683; b) A. T. Smith, R. Mueller, M. D. Toscano, P. Kast, H. W. Hellinga, D. Hilvert, K. N. Houk, J. Am. Chem. Soc. 2008, 130, 15361-15373.

[16] E. A. Loris, S. Panjikar, M. Ruppert, L. Barleben, M. Unger, H. Schuebel, J. Stoeckigt, ChemBiol. 2007, 14, 979-985.










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