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Oxygenases Get to Grips with Polypeptides
Structure
Previews Oxygenases Get to Grips with Polypeptides Timothy D.H. Bugg1,* 1Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK *Correspondence: [email protected] DOI 10.1016/j.str.2009.06.004
Hydroxylation of two proline residues in hypoxia inducible factor Hif-1a is a key step in the response to hypoxia. Here, Chowdury et al. (2009) elucidate the structural basis of this process by describing a structure for prolyl hydroxylase PHD2 bound to a peptide substrate. Non-heme iron-dependent oxygenase enzymes are involved in a wide variety of biochemical pathways, ranging from natural product biosynthesis, where they catalyze important reactions in b-lactam and glycopeptide antibiotic biosynthesis, to oxidative degradation pathways, where they catalyze hydroxylation and oxidative cleavage reactions in the breakdown of naturally occurring and xenobiotic metabolites. Over the last ten years, it has emerged that oxygenases play a key role in signaling pathways for oxygen sensing in mammals. The hypoxia-inducible factor HIF-1a, which regulates transcription of a set of genes under hypoxic conditions, is hydroxylated on two proline residues, Pro-564 and Pro-402, by a set of three 2-oxoglutarate-dependent prolyl hydroxylases (human PHD 1, 2, and 3), leading to ubiquitin-mediated degradation (Schofield and Ratcliffe, 2004). These hydroxylation steps respond directly to oxygen levels in cells, since oxygen is the substrate for hydroxylation, and therefore provide the link between oxygen concentration and cell signaling. The structural basis of peptide hydroxylation by the PHD family has been eagerly
awaited. The close relative of this group of enzymes is collagen prolyl hydroxylase, catalyzing the hydroxylation of multiple proline residues in the structural protein collagen, which has been studied for many years but has until recently eluded structure determination, in the form of an algal prolyl 4-hydroxylase enzyme (Koski et al., 2007). The structure of human PHD was determined in 2006, revealing that the active site iron (II) cofactor is ligated by a facial tridentate set of ligands (His-313, Asp-315, His-374) and is buried quite deeply in the active site pocket, explaining the formation of a tight complex with iron (II) and 2-oxoglutarate (McDonough et al., 2006). The facial tridentate His, His, Asp/Glu ligand set is found widely in non-heme iron-dependent enzymes (Koehntop et al., 2005), and allows binding of a bidentate substrate or cosubstrate (such as 2-oxoglutarate), and a sixth co-ordination site that can be used for dioxygen binding. In this issue, Chowdury et al. (2009) report the first structure of a complex between PHD2 and its substrate peptide CODD, revealing interactions between the enzyme and its substrate. In compar-
ison with the structure of ligand-free PHD2, a significant conformational change of the b2b3 loop is observed. The conformational change appears to be anchored by a salt bridge between Arg-252 and Asp-254, which can be targeted for the development of enzyme inhibitors. The structure also elegantly confirms recent stereo-chemical studies (Loenarz et al., 2009) that suggest that the conformation of the prolyl residue of the bound substrate is important for recognition and catalysis. The bound prolyl residue (corresponding to Pro-564 of HIF-1a) adopts a C4 endo conformation, in which the proR hydrogen at C-4 points directly toward the site where the reactive iron (IV) oxo intermediate will form during the catalytic mechanism (see Figure 1). In contrast, the product adopts a C4 exo conformation, in which the 4-hydroxyl group forms an unfavorable steric clash with nearby active site residues, thereby promoting product release. Unlike oxygenases that bind small molecule substrates, these prolyl hydroxylase enzymes are binding polypeptide substrates, which bring additional demands on the enzyme. The hydroxylation site must
Figure 1. Conformations of Prolyl Residue in the Substrate and Product of the PHD2 Catalyzed Reaction
Structure 17, July 15, 2009 ª2009 Elsevier Ltd All rights reserved 913
Structure
Previews be recognized and the polypeptide loop bound to the hydroxylase active site. This might require some local conformational changes in the polypeptide substrate, necessitating energy expenditure to overcome protein folding interactions. It therefore may be important that the PHD2 prolyl hydroxylase structure is flexible and dynamic, which was also observed in the structure of prolyl 4-hydroxylase (Koski et al., 2007). Similar issues are faced by protein kinase and phosphatase enzymes that catalyze phosphorylation and dephosphorylation reactions on polypeptide substrates. Protein tyrosine phosphatase 1B is known to undergo a conformational change of a surface loop upon binding of its peptide substrate (Jia et al., 1995), and it is known that inhibitors can bind to an allosteric site that blocks the mobility of this loop (Wiesmann et al., 2004). Structure determination of the Src protein kinase with or without bound inhibitors also shows
evidence of conformational changes in the protein structure (Breitenlechner et al., 2005). There are indications that 2-oxoglutarate-dependent oxygenases are involved in a variety of biochemical and signaling pathways in mammals and metazoans (Loenarz and Schofield, 2008). It may therefore prove to be the case that protein hydroxylation is a more widespread posttranslational modification reaction than previously thought.
REFERENCES
Jia, Z., Barford, D., Flint, A.J., and Tonks, N.K. (1995). Science 268, 1754–1758. Koski, M.K., Hieta, R., Bollner, C., Kivirikko, K.I., Myllyharju, J., and Wierenga, R.K. (2007). J. Biol. Chem. 282, 37112–37123. Koehntop, K.D., Emerson, J.P., and Que, L., Jr. (2005). J. Biol. Inorg. Chem. 10, 87–93. Loenarz, C., and Schofield, C.J. (2008). Nat. Chem. Biol. 4, 152–156. Loenarz, C., Mecinovic, J., Chowdury, R., McNeil, L.A., Flashman, E., and Schofield, C.J. (2009). Angew Chem. Int. Ed. Engl. 48, 1784–1787. McDonough, M.A., Li, V., Flashman, E., Chowdury, R., Mohr, C., Lienard, B.M., Zondlo, J., Oldham, N.J., Clifton, I.J., Lewis, J., et al. (2006). Proc. Natl. Acad. Sci. USA 103, 9814–9819.
Breitenlechner, C.B., Kairies, N.A., Honold, K., Scheiblich, S., Koll, H., Greiter, E., Koch, S., Schafer, W., Huber, R., and Engh, R.A. (2005). J. Mol. Biol. 353, 222–231.
Schofield, C.J., and Ratcliffe, P.J. (2004). Nat. Rev. Mol. Cell Biol. 5, 343–354.
Chowdury, R., McDonough, M.A., Mecinovic, J., Loenarz, C., Flashman, E., Hewitson, K.S., Domene, C., and Schofield, C.J. (2009). Structure 17, this issue, 981–989.
Wiesmann, C., Barr, K.J., Kung, J., Zhu, J., Erlanson, D.A., Shen, W., Fahr, B.J., Zhong, M., Taylor, L., Randal, M., et al. (2004). Nat. Struct. Mol. Biol. 11, 730–737.
KasA, Another Brick in the Mycobacterial Cell Wall Stephanie Swanson,1 Kuppan Gokulan,1 and James C. Sacchettini1,* 1Department of Chemistry, Texas A&M University, College Station, TX 77842, USA *Correspondence: [email protected] DOI 10.1016/j.str.2009.06.006
Mycobacterium tuberculosis, the causative agent of tuberculosis, a disease that has been plaguing humanity for centuries, has a unique cell wall composition believed to be critical for pathogenicity. Luckner et al. (2009) now describe the structure of KasA, an enzyme involved in cell wall biosynthesis. The mycobacterial cell wall contains an unusually complex, lipid-rich coat that has been proposed to help the pathogen survive the harsh environment of the macrophage and is thought to provide intrinsic resistance to common antibacterial agents. The most distinctive feature of the cell wall is the presence of very long chain (C60-90) a-branched, b-hydroxy fatty acids, known as mycolic acids. In the mycobacterial cell wall, mycolic acids exist in the form of trehalose dimycolate (TDM or chord factor), or they are esterified to the nonreducing termini of the arabinogalactan-peptidoglycan complex (Brennan and Nikaido, 1995).
In M. tuberculosis, the four catalytic steps of the de novo synthesis of fatty acids are achieved by a eukaryotic-like, so-called type I fatty acid synthase (FAS-I) system. The FAS-I system is composed of two very long polypeptides (a 640 kDa functional homo-dimer), which has multiple active centers. Further elongation of the fatty acids to mycolic acids is carried out by the type II FAS-II system. The FAS-II system uses monofunctional enzymes to carry out each step of the reaction, either separately or as a multienzyme complex. The mycolic acid FAS-II enzymes are very similar to the fatty acid biosynthetic enzymes found in other bacteria and
914 Structure 17, July 15, 2009 ª2009 Elsevier Ltd All rights reserved
plants. Indeed, mycobacteria are unusual in that they have both the FAS-I and FAS-II systems. The mycobacterial FAS-II system can only elongate a C16-C20-acyl chain into a mixture of mycolic acids that range from C54 to C63. KasA belongs to the FAS-II system and employs FAS-I products as starter molecules to synthesize meromycolates, fatty acids with C50 chain lengths or longer that are intermediates in mycolic acid production, by a condensation reaction using malonylACPM. Another interesting aspect of the mycobacterial mycolic acid biosynthetic pathway is the presence of three homologous condensing enzymes in the FAS-II
Previews Oxygenases Get to Grips with Polypeptides Timothy D.H. Bugg1,* 1Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK *Correspondence: [email protected] DOI 10.1016/j.str.2009.06.004
Hydroxylation of two proline residues in hypoxia inducible factor Hif-1a is a key step in the response to hypoxia. Here, Chowdury et al. (2009) elucidate the structural basis of this process by describing a structure for prolyl hydroxylase PHD2 bound to a peptide substrate. Non-heme iron-dependent oxygenase enzymes are involved in a wide variety of biochemical pathways, ranging from natural product biosynthesis, where they catalyze important reactions in b-lactam and glycopeptide antibiotic biosynthesis, to oxidative degradation pathways, where they catalyze hydroxylation and oxidative cleavage reactions in the breakdown of naturally occurring and xenobiotic metabolites. Over the last ten years, it has emerged that oxygenases play a key role in signaling pathways for oxygen sensing in mammals. The hypoxia-inducible factor HIF-1a, which regulates transcription of a set of genes under hypoxic conditions, is hydroxylated on two proline residues, Pro-564 and Pro-402, by a set of three 2-oxoglutarate-dependent prolyl hydroxylases (human PHD 1, 2, and 3), leading to ubiquitin-mediated degradation (Schofield and Ratcliffe, 2004). These hydroxylation steps respond directly to oxygen levels in cells, since oxygen is the substrate for hydroxylation, and therefore provide the link between oxygen concentration and cell signaling. The structural basis of peptide hydroxylation by the PHD family has been eagerly
awaited. The close relative of this group of enzymes is collagen prolyl hydroxylase, catalyzing the hydroxylation of multiple proline residues in the structural protein collagen, which has been studied for many years but has until recently eluded structure determination, in the form of an algal prolyl 4-hydroxylase enzyme (Koski et al., 2007). The structure of human PHD was determined in 2006, revealing that the active site iron (II) cofactor is ligated by a facial tridentate set of ligands (His-313, Asp-315, His-374) and is buried quite deeply in the active site pocket, explaining the formation of a tight complex with iron (II) and 2-oxoglutarate (McDonough et al., 2006). The facial tridentate His, His, Asp/Glu ligand set is found widely in non-heme iron-dependent enzymes (Koehntop et al., 2005), and allows binding of a bidentate substrate or cosubstrate (such as 2-oxoglutarate), and a sixth co-ordination site that can be used for dioxygen binding. In this issue, Chowdury et al. (2009) report the first structure of a complex between PHD2 and its substrate peptide CODD, revealing interactions between the enzyme and its substrate. In compar-
ison with the structure of ligand-free PHD2, a significant conformational change of the b2b3 loop is observed. The conformational change appears to be anchored by a salt bridge between Arg-252 and Asp-254, which can be targeted for the development of enzyme inhibitors. The structure also elegantly confirms recent stereo-chemical studies (Loenarz et al., 2009) that suggest that the conformation of the prolyl residue of the bound substrate is important for recognition and catalysis. The bound prolyl residue (corresponding to Pro-564 of HIF-1a) adopts a C4 endo conformation, in which the proR hydrogen at C-4 points directly toward the site where the reactive iron (IV) oxo intermediate will form during the catalytic mechanism (see Figure 1). In contrast, the product adopts a C4 exo conformation, in which the 4-hydroxyl group forms an unfavorable steric clash with nearby active site residues, thereby promoting product release. Unlike oxygenases that bind small molecule substrates, these prolyl hydroxylase enzymes are binding polypeptide substrates, which bring additional demands on the enzyme. The hydroxylation site must
Figure 1. Conformations of Prolyl Residue in the Substrate and Product of the PHD2 Catalyzed Reaction
Structure 17, July 15, 2009 ª2009 Elsevier Ltd All rights reserved 913
Structure
Previews be recognized and the polypeptide loop bound to the hydroxylase active site. This might require some local conformational changes in the polypeptide substrate, necessitating energy expenditure to overcome protein folding interactions. It therefore may be important that the PHD2 prolyl hydroxylase structure is flexible and dynamic, which was also observed in the structure of prolyl 4-hydroxylase (Koski et al., 2007). Similar issues are faced by protein kinase and phosphatase enzymes that catalyze phosphorylation and dephosphorylation reactions on polypeptide substrates. Protein tyrosine phosphatase 1B is known to undergo a conformational change of a surface loop upon binding of its peptide substrate (Jia et al., 1995), and it is known that inhibitors can bind to an allosteric site that blocks the mobility of this loop (Wiesmann et al., 2004). Structure determination of the Src protein kinase with or without bound inhibitors also shows
evidence of conformational changes in the protein structure (Breitenlechner et al., 2005). There are indications that 2-oxoglutarate-dependent oxygenases are involved in a variety of biochemical and signaling pathways in mammals and metazoans (Loenarz and Schofield, 2008). It may therefore prove to be the case that protein hydroxylation is a more widespread posttranslational modification reaction than previously thought.
REFERENCES
Jia, Z., Barford, D., Flint, A.J., and Tonks, N.K. (1995). Science 268, 1754–1758. Koski, M.K., Hieta, R., Bollner, C., Kivirikko, K.I., Myllyharju, J., and Wierenga, R.K. (2007). J. Biol. Chem. 282, 37112–37123. Koehntop, K.D., Emerson, J.P., and Que, L., Jr. (2005). J. Biol. Inorg. Chem. 10, 87–93. Loenarz, C., and Schofield, C.J. (2008). Nat. Chem. Biol. 4, 152–156. Loenarz, C., Mecinovic, J., Chowdury, R., McNeil, L.A., Flashman, E., and Schofield, C.J. (2009). Angew Chem. Int. Ed. Engl. 48, 1784–1787. McDonough, M.A., Li, V., Flashman, E., Chowdury, R., Mohr, C., Lienard, B.M., Zondlo, J., Oldham, N.J., Clifton, I.J., Lewis, J., et al. (2006). Proc. Natl. Acad. Sci. USA 103, 9814–9819.
Breitenlechner, C.B., Kairies, N.A., Honold, K., Scheiblich, S., Koll, H., Greiter, E., Koch, S., Schafer, W., Huber, R., and Engh, R.A. (2005). J. Mol. Biol. 353, 222–231.
Schofield, C.J., and Ratcliffe, P.J. (2004). Nat. Rev. Mol. Cell Biol. 5, 343–354.
Chowdury, R., McDonough, M.A., Mecinovic, J., Loenarz, C., Flashman, E., Hewitson, K.S., Domene, C., and Schofield, C.J. (2009). Structure 17, this issue, 981–989.
Wiesmann, C., Barr, K.J., Kung, J., Zhu, J., Erlanson, D.A., Shen, W., Fahr, B.J., Zhong, M., Taylor, L., Randal, M., et al. (2004). Nat. Struct. Mol. Biol. 11, 730–737.
KasA, Another Brick in the Mycobacterial Cell Wall Stephanie Swanson,1 Kuppan Gokulan,1 and James C. Sacchettini1,* 1Department of Chemistry, Texas A&M University, College Station, TX 77842, USA *Correspondence: [email protected] DOI 10.1016/j.str.2009.06.006
Mycobacterium tuberculosis, the causative agent of tuberculosis, a disease that has been plaguing humanity for centuries, has a unique cell wall composition believed to be critical for pathogenicity. Luckner et al. (2009) now describe the structure of KasA, an enzyme involved in cell wall biosynthesis. The mycobacterial cell wall contains an unusually complex, lipid-rich coat that has been proposed to help the pathogen survive the harsh environment of the macrophage and is thought to provide intrinsic resistance to common antibacterial agents. The most distinctive feature of the cell wall is the presence of very long chain (C60-90) a-branched, b-hydroxy fatty acids, known as mycolic acids. In the mycobacterial cell wall, mycolic acids exist in the form of trehalose dimycolate (TDM or chord factor), or they are esterified to the nonreducing termini of the arabinogalactan-peptidoglycan complex (Brennan and Nikaido, 1995).
In M. tuberculosis, the four catalytic steps of the de novo synthesis of fatty acids are achieved by a eukaryotic-like, so-called type I fatty acid synthase (FAS-I) system. The FAS-I system is composed of two very long polypeptides (a 640 kDa functional homo-dimer), which has multiple active centers. Further elongation of the fatty acids to mycolic acids is carried out by the type II FAS-II system. The FAS-II system uses monofunctional enzymes to carry out each step of the reaction, either separately or as a multienzyme complex. The mycolic acid FAS-II enzymes are very similar to the fatty acid biosynthetic enzymes found in other bacteria and
914 Structure 17, July 15, 2009 ª2009 Elsevier Ltd All rights reserved
plants. Indeed, mycobacteria are unusual in that they have both the FAS-I and FAS-II systems. The mycobacterial FAS-II system can only elongate a C16-C20-acyl chain into a mixture of mycolic acids that range from C54 to C63. KasA belongs to the FAS-II system and employs FAS-I products as starter molecules to synthesize meromycolates, fatty acids with C50 chain lengths or longer that are intermediates in mycolic acid production, by a condensation reaction using malonylACPM. Another interesting aspect of the mycobacterial mycolic acid biosynthetic pathway is the presence of three homologous condensing enzymes in the FAS-II