Structure-Function Relationships in Serine Hydroxymethyltransferase

Bhavani, B. S. (2009) Structure-Function Relationships in Serine Hydroxymethyltransferase. PhD thesis, University of Mysore.

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Abstract

A study of enzymes is central to an understanding of biological function. The binding of substrate(s) to an enzyme and its fit at the active site facilitates a multitude of chemical reactions. The mechanisms of catalysis include general acid-base, covalent and metal ion catalysis. The study of structure-function of enzymes has been central to the elucidation of catalytic mechanism of biochemical reactions. In addition to the factors mentioned above, coenzymes which are vitamin derivatives have provided several new insights into biology. The versatility of vitamin B6 and the wide distribution of pyridoxal 5’-phosphate (PLP)-dependent enzymes are reflected in the observation that 4% of all catalytic reactions involve this co-enzyme. The availability of biochemical and structural information on more than 140 PLP-enzymes gives a good handle to understand the organization of PLP-enzymes in detail. This information has helped in elucidating the diverse reaction mechanisms of PLP-enzymes. SHMT, one of the PLP-enzymes, the subject of this study belongs to the α-family. It links amino acid and nucleotide metabolism. Serine hydroxymethyltransferase (SHMT) catalyzes THF-dependent hydroxymethyltransfer from L-Ser to tetrahydrofolate (THF) to yield 5, 10-CH2 THF and Gly. This reaction provides one-carbon fragments for a wide variety of end products in mammalian systems. SHMT being a part of thymidylate cycle, suggested that it could be an alternative target for cancer chemotherapy. SHMT, in addition to L-Ser and Gly inter-conversion, catalyzes THF-independent cleavage of 3-hydroxy amino acids. L-Thr/L-allo Thr cleavage by SHMT results in production of acetaldehyde which is a major flavoring compound in production of fermented dairy products. Hence SHMT can be used as a starter culture in the manufacture of dairy products. SHMT can also be used as a biocatalyst in the synthesis of β-hydroxy-α-amino acid derivatives. 2 The objectives of the present investigation are: biochemical characterization of selected residues involved in THF-dependent and -independent reactions; crystallization of the mutant enzymes with their substrate(s)/inhibitor complexes to understand the role of these residues; probe the retroaldol and direct displacement mechanisms for the THFdependent L-Ser cleavage; establish the mechanism of THF-independent cleavage of 3-hydroxy amino acids by mutation of specific amino acid residues; and study the interaction of chemical inhibitors and compounds from natural sources to understand the role of SHMT in cancer. With these objectives the present investigation was undertaken and results and conclusions are presented in the form of thesis entitled “Structure – Function Relationships in Serine Hydroxymethyltransferase”. The present study is divided into four chapters: Chapter 1: The role of lysine226 in the reaction catalyzed by bsSHMT Lys residue at the active site of PLP-enzymes in addition to anchoring PLP functions as a proton acceptor or a donor in catalysis. It has been proposed that Lys 229 in eSHMT is crucial for product expulsion, which is a rate determining step of catalysis. Lys 226 of bsSHMT was mutated to Met and Gln, overexpressed and the mutant enzymes were purified. The mutant enzymes contained 1 mol of PLP per mol of subunit suggesting that Schiff’s base formation with Lys was not essential for PLP binding. K226M and K226Q bsSHMT were inactive for THF-dependent cleavage of L-Ser. However, cleavage of L-allo Thr and transamination reaction was not abolished completely. K226M bsSHMT had distinct absorbance maximum at 412 nm and K226Q bsSHMT was similar to that of bsSHMT. The crystal structure of K226M bsSHMT revealed that PLP was bound at the active site in an orientation different (16°) from that of the wild-type enzyme. The absence of reaction intermediate at 388 nm on interaction with methoxyamine (MA) corroborates the suggestion that the active site of K226M bsSHMT was different from that of bsSHMT. Both the Lys mutants were capable of forming an external aldimine; this was also supported by the crystal structure of K226M-Ser/Gly complexes. Spectral studies show the formation of a small amount of quinonoid intermediate on addition of Gly and THF/FTHF to K226M bsSHMT. However, stopped-flow studies suggested enhanced quinonoid intermediate formed on addition of THF was affected drastically. In SHMT, formation of an external 3 aldimine is accompanied by the change in orientation of PLP by 25°. The orientation of PLP in the external aldimine form (25°) changes to 16° during quinonoid intermediate formation. The quinonoid intermediate is stabilized by interactions of Lys at the active site. In the absence of the -NH2 group of Lys, the conversion of the external aldimine to product quinonoid intermediate, that is, the change in orientation of PLP from 25° to 16°, may not be possible. This in turn could lead to shifting of equilibrium towards the substrate external aldimine form (L-Ser form). These results show that Lys 226 is responsible for flipping of PLP from one orientation to another, which is accompanied by Cα-Cβ bond cleavage. This flip is important in the THF-mediated enhanced Cα proton abstraction from Gly in the reverse reaction. Chapter 2: The involvement of glutamate 53 in binding of L-serine and folate, and conversion of bsSHMT from ‘open’ to ‘closed’ form An examination of the crystal structure of bsSHMT binary and ternary complexes suggested that E53 interacts with L-Ser and FTHF. Glu 53 was mutated to Gln and structural and biochemical studies were carried out to examine the role of this residue in catalysis. The mutant enzyme was completely inactive for THF-dependent cleavage of L-Ser, whereas there was a 1.5-fold increase in the rate of THF-independent reaction with L-allo Thr. Spectral studies showed that E53Q bsSHMT had absorbance maximum at 425 nm and the addition of L-Ser/Gly resulted in formation of an external aldimine. The crystal structure of E53Q bsSHMT was similar to that of the wild-type enzyme, except for significant changes at Q53, Y60 and Y61. E53Q bsSHMT binary complex with L-Ser or Gly showed that the side chain of L-Ser and carboxyl of Gly were in two conformations in the respective external aldimine structures. The loss in characteristic decrease in molar ellipticity on addition of L-Ser and loss of enhanced thermal stability suggested that E53Q bsSHMT was unable to undergo a conformational change from ‘open’ to ‘closed’ form in which THF-dependent reaction occurs. Addition of THF/FTHF to E53Q bsSHMT-Gly complex showed the formation of a quinonoid intermediate. Stopped-flow studies were performed to obtain rate constants for the formation of quinonoid intermediate with mutant and wild-type enzymes. However the quinonoid intermediate formed by the mutant enzyme 4 was unstable. Dialysis experiments and dissociation constants for FTHF suggested that, the affinity for FTHF to mutant binary complex was lower than bsSHMT. This could be due to loss of interaction of N10 and formyl oxygen of FTHF the enzyme. These results suggested that Glu plays an important role in folate binding. The crystal structure of the complex obtained on co-crystallization of E53Q bsSHMT with Gly and FTHF revealed that it exists in a gem-diamine form with an orientation of PLP similar to that of wild-type ternary complex. However, electron density for FTHF was not observed. The formation of gem-diamine in the above conditions was supported by circular dichroism measurements of E53Q bsSHMT ternary complex in the visible region. The absence of FTHF in the crystal structure and the formation of quinonoid intermediate suggest that there was an initial binding of FTHF to the binary complex of E53Q bsSHMT leading to an alteration in the orientation of PLP. Subsequently, FTHF falls off from the active site leaving behind the gem-diamine complex. The differences between the structures of this complex and Gly external aldimine suggest that the changes induced by initial binding of FTHF are retained, even though FTHF was absent in the final structure. These observations indicate that mutant enzyme exhibits a phenomenon known as “enzyme memory”. In this concept, binding of ligands caused conformational changes in the enzyme and even after removal of ligands such imprints of ligand binding were retained by the enzyme. Double reciprocal plots of bsSHMT ternary complex and E53Q bsSHMT-Gly-(FTHF) complex, supports the suggestion that, mutant enzyme exhibits ‘enzyme memory’. There are not many examples of structural evidence for ‘enzyme memory’. The results obtained from these studies suggest that E53 plays an essential role in THF⁄FTHF binding and in the proper positioning of Cβ of L-Ser for direct attack by N5 of THF. It does not have an important role in THF-independent reactions. 5 Chapter 3: The role of tyrosine residues in cofactor binding and elucidation of mechanism for the tetrahydrofolate-independent cleavage of L-allo threonine Tyr residues play multiple functions in enzyme catalysis. The crystal structure of bsSHMT showed that hydroxyl group of Y51 interacted with the phosphate group of PLP. Binary complex of bsSHMT-Ser showed change in conformation in Y61 on binding of L-Ser. The new conformation Y61 is close to Cβ of the bound ligand, L-Ser. The active site Tyr’s was mutated to Y51F, Y61F and Y61A bsSHMT to understand their role in bsSHMT catalysis. Mutation of these residues resulted in a complete loss of THF-dependent and - independent activities. The PLP content of Y51F and Y61F bsSHMT as isolated was 0.2 mol/mol and 0.6 mol/mol of subunit, respectively, compared to 1 mol/mol of subunit in bsSHMT. The mutant enzyme could be completely reconstituted with PLP. However, there was an alteration in the λmax value of the internal aldimine (396 nm) in the case of Y51F bsSHMT. A decrease in the rate of reduction with NaCNBH3 and a loss of the intermediate in the interaction with MA suggests that the active site environment is altered in the case of Y51F bsSHMT due to mutation. The mutation of Y51 to F strongly affects the binding of PLP, possibly as a consequence of a change in the orientation of the phenyl ring (75°) in Y51F bsSHMT structure. The results obtained for Y61F were also similar to that of Y51F bsSHMT. Y61A bsSHMT, as isolated had 1mol/mol of subunit and a absorption maximum of 425 nm similar to bsSHMT. In addition, Y61A bsSHMT was able to form an external aldimine; this change was supported by enhanced thermal stability of the mutant enzyme on addition of L-Ser. However, the formation of the quinonoid intermediate was hindered. An examination of the active site geometry of bsSHMT and Y51F and Y61A mutants show that Y51 and Y61 are not suitably placed for the removal of the proton from the hydroxyl group of L-allo Thr. In bsSHMT, the hydroxyl group of Y51 is at a distance of 3.6 Å and 3.8 Å from Cα of Gly and Ser, respectively. Of the two residues, Y51 is unlikely to be involved in proton abstraction from Cα of the bound ligand due to its longer distance and improper geometry. In contrast, the OH of Y61 is at a distance of 3.3 Å and 3.2 Å from Cα of Gly and Ser, respectively. In Gly, L-Ser and L-allo Thr complexes of Y51F bsSHMT, the OH of 6 Y61 and Cα of bound ligand are at distances 4.97, 4.39 and 4.39 Å, respectively. Y61 therefore may be involved in Cα proton abstraction in the THF-independent reaction. This might explain the loss of L-allo Thr cleavage activity of Y51F bsSHMT. It could therefore, be concluded that Y51 is important for PLP binding and proper positioning of Y61, while Y61 could be involved in the abstraction of the proton from Cα carbon of L-allo Thr. Based on these observations, a possible mechanism for SHMT catalyzed cleavage of L-allo Thr is suggested. Chapter 4: The interaction of bsSHMT with specific inhibitors from extracts of various spices The pivotal role of SHMT in the interconversion of folate coenzymes and its altered kinetic properties in neoplastic tissues suggested that it could be a potential target for cancer chemotherapy. This chapter deals with understanding the interaction of aminooxy compounds with K226M, K226Q, E53Q, Y51F, Y61F and Y61A bsSHMT. Methoxyamine (MA) and aminooxyacetic acid (AAA) interacted with the mutant proteins in different manner. K226M, K226Q bsSHMT did not react with MA and E53Q, Y51F, Y61A and Y61F bsSHMT failed to form 388 nm intermediate on addition of MA. These results suggest that any change at the active site environment or PLP orientation in bsSHMT could lead to the loss of intermediate formation on interaction with MA. Lys mutants were able to interact with AAA, however the interaction rate was slower compared to bsSHMT suggesting that AAA was a more reactive compared to MA. The absence of Schiff’s base and a change in the orientation of PLP at the active site of K226M and K226Q bsSHMTs probably results in poor binding of AAA at the active site. Y51F, Y61F and Y61A bsSHMT showed a similar interaction pattern as that of bsSHMT. Only E53Q bsSHMT showed the formation of an intermediate absorbing at 388 nm on addition of AAA. Stopped-flow studies suggested a drastic decrease in the rate at which AAA interacts with E53Q bsSHMT, when compared to bsSHMT. It is possible that Glu 53 may be the crucial residue involved in binding of AAA as well as in enhancing the rate of the reaction. 7 A fruitful approach of identifying compounds which have anti-carcinogenic effect is to carry out homology modeling and docking of compounds available in several chemical libraries. Homology modeling and docking of commercially available folate analogues were performed. Docking of folate analogues resulted in identification of 14 best compounds of these four water soluble compounds were examined for their ability to inhibit bsSHMT. Activity studies suggested that folate analogues failed to inhibit bsSHMT. In addition to synthetic compounds, naturally occurring compounds from spices that can potentially work as anti-cancer agents were also analyzed for bsSHMT inhibition. The effect of spices such as garlic, ginger, chilli, turmeric extracts was examined. Among all spices, heat-treated and lyophilized garlic extract showed 91% inhibition of bsSHMT. D and L-allyl Gly analogues of substrate L-Ser and S-allyl Cys (probable inhibitor form garlic extract) D and L-allyl Gly, Alliin did not inhibit bsSHMT activity. Docking studies with S-allyl Cys, D and L-allyl Gly and alliin suggest that alliin, L-allyl Gly and S-allyl Cys bind at the active site of bsSHMT with different orientations. However, alliin and L-allyl Gly bind in similar orientation/position which is different from that of OADS. Binding of S-allyl Cys was almost similar to that of OADS. These results suggest that S-allyl Cys could be a potential inhibitor. Chemical synthesis of S-allyl Cys and further activity measurement has to be carried out to demonstrate the inhibition. These results emphasize the importance of examining the naturally occurring and synthetic compounds as possible chemotherapeutic agents. The results presented in this study bring in the importance of correlating structural information with catalytic function. The mutation of Lys 226 of bsSHMT demonstrated its role in facilitating the change in orientation of PLP during catalysis. Glu 53 on interaction with L-Ser positions the Cα-Cβ bond for attack by N5 of THF, a crucial step in L-Ser cleavage. The loss of efficient binding of FTHF to E53Q-Gly binary complex up on Glu 53 mutation results in the enzyme exhibiting a phenomenon called ‘enzyme memory’. Tyr 51 in involved in cofactor binding. Tyr 61 plays an important role in Cα proton abstraction leading to THF-independent cleavage of 3-hydroxy amino acids. These observations prompted proposal of a new mechanism for aldol cleavage of 3-hydroxy amino acids. Both 8 commercially available and naturally occurring bioactive compounds from spices that can serve as anti-cancer agents were analyzed for bsSHMT inhibition. Garlic extract showed the maximum inhibition suggesting the importance of the naturally occurring compounds as possible and potential chemotherapeutic agents against cancer. The summary and conclusions in the thesis briefly highlights the salient features of the present investigation. The literature sited in the complete text is arranged in alphabetical order under reference section, which gives all the relevant details including title, journal year, volume and pagination.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	enzymes, THF-independent cleavage, biochemical characterization
Subjects:	600 Technology > 08 Food technology > 16 Nutritive value > 05 Enzymes 500 Natural Sciences and Mathematics > 07 Life Sciences > 03 Biochemistry & Molecular Biology
Divisions:	Protein Chemistry and Technology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	01 Mar 2011 04:30
Last Modified:	28 Dec 2011 10:21
URI:	http://ir.cftri.res.in/id/eprint/9937

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