Prof. Rik K. Wierenga, Ph.D.
Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu
C-P4H hydroxylates prolines of proline-rich peptides. The α-chain has three domains of which the N-terminal domain is important for forming the α2-dimer, the middle domain is the peptide-substrate-binding (PSB) domain and the C-terminal domain is the catalytic domain (CAT). The CAT domain provides the active site. The in vivo C-P4H substrate is procollagen. Procollagen includes a polypeptide of about 300 (XYG)-repeats of which the X and Y residues are very often prolines and in the mature collagen the Y-proline is very often hydroxylated. What determines which PPG-triplet is hydroxylated? Is it determined by the PSB domain or by the CAT domain, or both? Our C-P4H research efforts are aimed at resolving the crystal structure of C-P4H, which will reveal the spatial location of the PSB domain with respect to the CAT domain and which will be critically important in understanding all aspects of the C-P4H reaction mechanism.
Our studies of the CoA dependent, thioester dependent enzymes are also aimed at understanding better the reaction mechanism of these enzymes. The chemistry of the reactions catalysed by these enzymes is qualitatively understood, but a quantitative understanding of the free energy profile of the reaction is rather incomplete. Figure 1 visualises schematically the free energy profile of the reaction catalysed by the Zoogloea ramigera biosynthetic thiolase, for which the rate limiting step in the degradative direction is the transfer of the acetyl group from the acetylated cysteine to CoA (Figure 1). In some other thiolases the rate limiting step of the degradative direction is the formation of the acetylated enzyme. In the active site of thiolases, and other β-oxidation enzymes, oxyanion holes (OAHs), stabilise the transition state structure, and therefore determine the free energy profile of the catalysed reactions. Our studies include achieving a better understanding of the role of these OAHs.
Figure 1. Simplified scheme of the free energy profile of the thiolase reaction (in the degradative direction) of the Zoogloea ramigera thiolase. Acetoacetyl-CoA (AcAcS-CoA) is converted into two molecules of acetyl-CoA (AcS-CoA), via a covalent intermediate (AcS-enzyme). The rate limiting step (indicated by a *) is the transfer of the acetyl group from the acetylated cysteine to CoA.
The major progress in 2016 in the C-P4H and TFE projects concerns the development of purification protocols of new variants. In the C-P4H project we now have access to human C-P4H-I as well as human C-P4H-II. For these purification protocols we use the CyDisCo strains developed by the Lloyd Ruddock group. In the TFE project we now have purification protocols for a member of each of the four TFE subfamilies, being Mycobacterium tuberculosis (MtTFE), aerobic E. coli TFE (EcTFE), anaerobic E. coli TFE (anEcTFE) and mitochondrial human TFE (HsTFE). Crystallisation studies with each of these variants are in progress. anEcTFE and HsTFE are membrane associated, which does provide a major additional challenge for obtaining sufficient amounts for structural biology research, including the crystallisation studies. Preliminary crystallization hits have been obtained for EcTFE and HsTFE in the sitting drop vapor-diffusion and the capillary crystallization method, respectively. For anEcTFE, negative stained EM images have been collected and analysed. Further optimization for cryoEM studies are underway. In addition, we calculated a low resolution model for the anEcTFE octamer by SAXS. For MtTFE, fragment screening approaches have been initiated at BESSY Berlin, where a number of data sets have been collected after soaking with various ligands and the data are being analysed currently. In addition, crystallographic binding studies with mutated variants of MtTFE are in progress. anEcTFE and HsTFE do not have a trans membrane helix, but are predicted to be anchored to one leaflet of the membrane bilayer. Such membrane anchoring has been characterised for the mitochondrial, very long chain acyl-CoA-dehydrogenase (VLCAD) (McAndrew et. al., 2008). VLCAD catalyses the first step of the β-oxidation pathway, meaning that its product is the substrate of the hydratase active site of TFE. Currently the membrane anchoring region of HsTFE is not known. It has been reported that the α-chain is anchored to the membrane and therefore mutagenesis studies of TFEa have been initiated to discover which regions could be responsible for this anchoring. Also the determination of the Michaelis-Menten constants for the substrates with acyl chains of four, ten and sixteen carbon atoms for each of the four TFEs is in progress. These compounds are provided to us by Dr. Werner Schmitz (University of Würzburg, Germany).
The catalytic site of thiolase contains two oxyanion holes, OAH1 and OAH2. The two hydrogen bond donors (HBDs) of OAH2 are always formed by main chain peptide NH-groups, whereas the HBDs of OAH1 are a water (of a water-Asn dyad) and a histidine side chain in the well studied thiolases. These thiolases are also known as the CNH-thiolases, which are characterised by three amino acid sequence fingerprints (CxS, NEAF, GHP) of three catalytic loops. The CxS-cysteine provides the nucleophilic cysteine, the NEAF-asparagine provides the asparagine side chain of the water-Asn dyad HBD of OAH1 and the NE2-atom of the GHP-histidine provides the second HBD of OAH1. Recently we solved crystal structures of the CHH-class of thiolases, being the leishmanial SCP2-thiolase (type-2), liganded with acetyl-CoA and acetoacetyl-CoA (Harijan et al., 2017). In this thiolase the corresponding fingerprints are CxS, HDCF and GHP. In this active site the HBDs of OAH1 are the NE2 atom of the HDCF histidine and the NE2 atom of the GHP histidine. An analysis of the hydrogen bond networks of the latter hydrogen bond donors suggest that the HDCF histidine is singly protonated and neutral, like the water-Asn dyad of the CNH-thiolases, and that the GHP-histidine is doubly protonated and positively charged (like in the CNH thiolases). The analysis of the available thiolase structures suggests that extended hydrogen bond networks are also important for the properties of the HBDs of OAH2. In Figure 2 it is shown that these HBDs are the NH-groups of peptide units of which the oxygen atoms are hydrogen bonded further, by which extensive hydrogen bond networks are formed. The importance of these hydrogen bond networks for the properties of the HBDs has also been studied in biocomputing studies (Guo and Salahub, 1998; Wieczorik and Dannenberg, 2003). In collaboration with the Andre Juffer group we study the importance for the thiolase reaction mechanism of the water molecules of these hydrogen bond networks.
Figure 2. Oxyanion hole-2 (OAH2) of the active site of the Zoogloea ramigera thiolase (PDB entry 1DM3). The thioester oxygen of the acetylated Cys89 is hydrogen bonded to N(Cys89) and N(Gly380). The oxygens of these peptide links are part of hydrogen bond networks. It is highlighted that the hydrogen bond network of O(Ile379) (of the Ile379-Gly380 peptide unit) extends further to Arg68 (from a neighboring subunit). Arg68 is a highly conserved arginine as found in an extensive multiple sequence alignment of 130 thiolase sequences.
We will continue our crystallisation attempts of human C-P4H-I and C-P4H-II. An intriguing question in this project concerns the function of the PSB domain and its possible importance for the substrate specificity and processivity properties (De Jong et al., 1991) of C-P4H. Crystallisation and ligand binding studies of the PSB domains of C-P4H-I, II, III have been initiated. Crystallisation studies are also proceeding with the various TFE variants and cryoEM studies will be continued. Studies of mitochondrial β-oxidation metabolism suggest substrate channeling, because intermediates of the b-oxidation pathway are not detected. For in vitro enzyme kinetic studies of TFE (Yao and Schulz, 1996) substrate channeling is observed. Our structural biology studies of MFE1 and TFE are also aimed at unravelling their substrate channeling mechanisms. The detailed reaction mechanism studies of the CoA dependent, thioester dependent enzymes are important to achieve better understanding of the free energy profile of the reaction cycle (Figure 1). Such knowledge is essential for developing non-natural enzymes harnessing the catalytic power of enzymes such that non-natural enzymes with tailor-made substrate and reaction specificity (Arnold, 2015) are obtained.
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Wieczorek R, Dannenberg JJ. H-bonding cooperativity and energetics of alpha-helix formation of five 17-amino acid peptides. J Am Chem Soc. 125:8124-8129, 2003.
Harijan RK, Mazet M, Kiema, TR, Bouyssou G, Alexson S, Bergmann U, Moreau P, Michels PAM, Bringaud F, Wierenga RK. The SCP2-thiolase-like protein (SLP) of Trypanosoma brucei is an enzyme involved in lipid metabolism. Proteins 84(8):1075-96, 2016.
Ithayaraja M, Janardan N, Wierenga RK, Savithri HS, Murthy MRN. Crystal structure of a thiolase from Escherichia coli at 1.8 Å resolution. Acta Crystallogr F Struct Biol Commun 72:534-544, 2016.
Krause M, Kiema TR, Neubauer P, Wierenga RK. Crystal structures of two monomeric TIM variants identified via a directed evolution protocol selecting for L-arabinose isomerase activity. Acta Crystallogr F Struct Biol Commun 72: 490-499, 2016.
Krause M, Wierenga RK. Towards new non-natural TIM-barrel enzymes using computational design and directed evolution approaches. In “Understanding enzymes: Function, Design, Engineering and Analysis”. Pan Stanford Publishing Pte. Ltd, Singapore, pp561-611, 2016.
Meriläinen G, Koski MK, Wierenga RK. The extended structure of the periplasmic region of CdsD, a structural protein of the type III secretion system of Chlamydia trachomatis. Protein Sci. 25: 987-998, 2016.
Richard JP, Amyes TL, Malabanan MM, Zhai X, Kim KJ, Reinhardt CJ, Wierenga RK, Drake EJ, Gulick AM. Structure-Function Studies on Hydrophobic Residues that Clamp a Basic Glutamate Side Chain During Catalysis by triosephosphate isomerase. Biochemistry 55:3036-3047, 2016.
Harijan RK, Kiema TR, Syed SM, Qadir I, Mazet M, Bringaud F, Michels PAM, Wierenga RK. Crystallographic substrate binding studies of Leishmania mexicana SCP2-thiolase (type-2): unique features of oxyanion hole-1. PEDS in press.
Rik Wierenga (University of Oulu)
Senior and Post-doctoral Investigators:
Rajaram Venkatesan (principal investigator, MCE, TFE, Academy of Finland)
Tiila-Riikka Kiema (senior postdoc, thiolases, IVDH, MFE1, maintenance of the diffraction unit, Biocenter Oulu)
Kristian Koski (senior postdoc, C-P4H, University of Oulu strategic funding targeted for BF operations)
Rajesh Harijan (postdoc, thiolases, PNP, Albert Einstein College of Medecine, New York)
Gabriele Cordara (postdoc, TIM, ECI2, Biocenter Oulu, Academy of Finland)
Sandhanakrishnan Cattavarayane (postdoc, MCE, Erkko Foundation)
Dhirendra Singh (postdoc, MCE, Erkko Foundation)
Abhinandan Murthy (PhD student, human C-P4H, University of Oulu)
Shiv Kumar Sah-Teli (PhD student, TFE, Academy of Finland)
Shruthi Sridhar (PhD student, MFE1, Biocenter Oulu)
Subhadra Dalwani (PhD student, TFE, Academy of Finland)
Walteri Tuompo (MD-PhD student, MCE, University of Oulu)
Ramita Sulu (P4H)
Chandan Tapa (thiolase)
Masud Julfiker (P4H)
Naveed Ahmad (MCE)
Anil Sohail (MCE)
Ramita Sulu (P4H)
Aleksi Sutinen (P4H)
Mohammed Tanvir Rahman (thiolase)
Mikko Hynönen (TFE)
Akke-Pekka Törmänen (TIM)
Ville Ratas (technician, crystallisation of proteins, Biocenter Oulu)
Ed Daniel (programmer, development of xtalPiMS, Diamond Light Source, UK)
Foreign Scientists: 17
BFSB Biocenter Finland Structural Biology network, chair person (till September 2016)
Instruct-FI Biocenter Finland Structural Biology network, vice-chair person (since September 2016)
Biostruct-X, EU, coordinator of the Oulu TID-centre
iNEXT, EU, coordinator of the Oulu TID-centre
BESSY, Berlin, Germany, member of the macromolecular crystallography beamline proposal evaluation panel
The EMBO biocatalysis conference: "The biochemistry and chemistry of biocatalysis: From understanding to design" in Oulu. June 12 till June 15, 2016, organiser
EU Projects (present and progress)
Last updated: 25/4/2017