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© 1999 Macmillan Magazines Ltd is unable to find the source, whereas just 4 nanograms elicits no response. In similar pheromone tests, splendipherin does not elicit a response from male L. splendida or either males or females of the closely related tree frog L. caerulea. We conclude that the aquatic, male sex pheromone splendipherin is therefore species specific. Paul A. Wabnitz*, John H. Bowie*, Michael J. Tyler†, John C. Wallace‡, Ben P. Smith† Departments of *Chemistry, †Environmental Biology and ‡Biochemistry, University of Adelaide, South Australia 5005, Australia 1. Johnstone, R. E. in Pheromones and Reproduction in Mammals (ed. Vandenbergh, J. G.) 8–17 (Academic, New York, 1983). 2. Butenandt, A. & Hecher, E. Z. in Techniques in Pheromone Research (eds Hummel, H. E. & Miller, T. E.) 4–23 (Springer, New York, 1984). 3. Agosta, W. C. J. Chem. Educat. 71, 242–254 (1994). 4. Houck, L. D. Am. Zool. 38, 108–117 (1998). 5. Lazarus, L. H. & Attila, M. Prog. Neurobiol. 41, 473–507 (1993). 6. Erspamer, V. Amphibian Biology (ed. Heatwole, H.) 178–350 (Surrey Beatty, Chipping Norton, New South Wales, Australia, 1993). 7. Wong, H., Bowie, J. H. & Carver, J. A. Eur. J. Biochem. 247, 545–557 (1997). 8. Steinborner, S. T., Currie, G. J., Bowie, J. H., Wallace, J. C. & Tyler, M. J. J. Peptide Res. 51, 121–126 (1998). 9. Birch, M. C. in Pheromones Vol. 32 (eds Neuberger, A. & Tatum, E. L.) 1–14 (North Holland, Amsterdam, 1974). 10. Kikuyama, S. et al. Science 267, 1643–1645 (1995). 11.Tyler, M. J., Stone, D. J. M. & Bowie, J. H. J. Pharmacol. Toxicol. Methods 28, 199–200 (1992). 12.Wabnitz, P. A., Walter, H., Tyler, M. J., Wallace, J. C. & Bowie, J. H. J. Peptide Res. 52, 477–483 (1998). oxygen atom. If this is the case, then DHP represents a new class of enzyme: a dehaloperoxygenase. Its mechanism of action is probably similar to that of other peroxidases, given the important role of the high-valency iron–oxo intermediate that is formed when hydrogen peroxide is added to the enzyme and the subsequent het- erolytic cleavage of the O–O bond 3 . The ability of peroxidases to form such an inter- mediate, which globins cannot, is consistent with the idea 4 that the proximal histidine and polar residues in the distal pocket are crucial. Peroxidases form a strong hydrogen bond between the N d1 nitrogen atom of the proximal histidine and a nearby glutamate, so the proximal histidine takes on a partial histidinate character. In contrast, the proxi- mal pocket of globins lacks a glutamate, so they form only a weak hydrogen bond 4 . In DHP, a hydrogen bond forms that is stronger than in globins but, rather than bonding to a carboxylate, it is generated by a roughly 60° rotation of the imidazole ring. Its hydrogen points directly into the oxygen atom of the peptide carbonyl group of the leucine at residue 83. The hydrogen bond is shorter than that of myoglobin, in which it is bifurcated (Fig. 1). The reorientation of the axial ligand is facilitated by a shift of the proximal histidine in the sequence by two residues. In peroxidases, the distal histidine func- tions as an acid or base in the transfer of brief communications NATURE | VOL 401 | 30 SEPTEMBER 1999 | www.nature.com 445 protons to the leaving water molecule, and the developing negative charge is stabilized by an arginine side chain 4 . This arrange- ment is possible because peroxidases do not use the distal cavity as an organic-substrate- binding pocket, as the reaction takes place at the haem edge 5 . We propose that DHP binds peroxide and uses the distal histidine to cleave the O–O bond. When the high-valency iron–oxo intermediate is ready, the distal histidine swings out of the cavity, enabling the substrate to enter the distal pocket and undergo oxidation. This hypothesis is sup- ported by the observation that the distal histidine in the native DHP is disordered between two positions, with one outside the distal pocket and the other inside (Fig. 1), whereas it is outside the pocket in the com- plex with 4-iodophenol. The distance between the N e2 atom of the distal histidine and the haem iron in globins is 4.1 to 4.6 Å, whereas in peroxi- dases it is 5.5 to 6.0 Å (ref. 6). In DHP, this distance is 5.4 Å for the position inside the distal cavity, which is related to a 2.4 Å shift of its a-carbon compared with myoglobin. The shift appears to be generated by the replacement of the glycine that follows the distal histidine in myoglobin by threonine in DHP. DHP serves primarily to dehalogenate biologically generated bromophenols, but it can also dehalogenate trichlorophenols and other anthropogenic pollutants. The effi- ciency of the enzyme is not significantly affected by the position and number of halogen atoms at the phenol ring 2 . The enzyme simply binds its organic substrate close to a highly reactive oxygen atom and so it does not need a system involving hydrogen bonds and other interactions that are features of enzymes with more sophisti- cated mechanisms. This mechanism means that its specificity can be altered by modify- ing the residues lining the distal cavity, making it potentially useful as a tool for bioremediation projects. Lukasz Lebioda*, Michael W. LaCount*, Erli Zhang*†, Yung Pin Chen‡, Kaiping Han‡, Margaret M. Whitton†, David E. Lincoln‡, Sarah A. Woodin‡ Departments of *Chemistry and ‡Biochemistry and Biological Sciences, University of South Carolina, Columbia, South Carolina 29208, USA e-mail: [email protected] †Parke-Davis Pharmaceutical Research, Division of Warner-Lambert, Ann Arbor, Michigan 48105, USA 1. Woodin, S. A., Marinelli, R. L. & Lincoln, D. E. J. Chem. Ecol. 19, 517–530 (1993). 2. Chen, Y. P., Woodin, S. A., Lincoln, D. E. & Lovell, C. R. J. Biol. Chem. 271, 4609–4612 (1996). 3. Dawson, J. H. Science 240, 433–439 (1988). 4. Poulos, T. L. Adv. Inorg. Biochem. 7, 1–36 (1988). 5. Ator, M. A. & Ortiz de Montellano, P. R. J. Biol. Chem. 262, 1542–1551 (1987). 6. Matsui, T., Ozaki, S., Liong, E., Phillips, G. N. & Watanabe, Y. J. Biol. Chem. 274, 2838–2844 (1999). Figure 1 The least-squares superposition of the haem groups of dehaloperoxidase (green) and myoglobin (Protein Data Bank entry code, 1mbo), showing the most important differences in their environment. The position of the distal histidine of dehaloper- oxidase out of the binding pocket is shown in cyan, and myoglobin residues are purple. Protein structure An enzymatic globin from a marine worm Some marine worms, such as Thelepus cris- pus and Notomastus lobatus, secrete bromi- nated aromatic molecules and other halogenated metabolites as repellants 1 . Other species, such as Amphitrite ornata, do not produce repellants but are adapted to the chemical warfare of N. lobatus and cohabit with them in estuarine mudflats. The halocompounds are tolerated by A. ornata as they are degraded by dehaloper- oxidase (DHP) 2 . We have determined the amino-acid sequence and crystal structure of DHP and find that its fold is typical of the globin family, indicating that the enzyme evolved from an oxygen carrier protein. Residues at the dimer interface do not correspond to those in tetrameric and dimeric haemoglobins and the spatial arrangement of the dimers is different. The complete amino-acid sequence is most sim- ilar to that of myoglobin from the sea hare, with 20.6% identity among 126 overlapping amino acids. The halophenols bind in the distal pocket of DHP, indicating that DHP removes the halogen by directly attacking an activated
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© 1999 Macmillan Magazines Ltd
is unable to find the source, whereas just 4nanograms elicits no response. In similarpheromone tests, splendipherin does notelicit a response from male L. splendida oreither males or females of the closely relatedtree frog L. caerulea. We conclude that theaquatic, male sex pheromone splendipherinis therefore species specific.Paul A. Wabnitz*, John H. Bowie*, Michael J. Tyler†, John C. Wallace‡, Ben P. Smith†Departments of *Chemistry, †EnvironmentalBiology and ‡Biochemistry, University of Adelaide, South Australia 5005, Australia
1. Johnstone, R. E. in Pheromones and Reproduction in Mammals
(ed. Vandenbergh, J. G.) 8–17 (Academic, New York, 1983).
2. Butenandt, A. & Hecher, E. Z. in Techniques in Pheromone
Research (eds Hummel, H. E. & Miller, T. E.) 4–23 (Springer,
New York, 1984).
3. Agosta, W. C. J. Chem. Educat. 71, 242–254 (1994).
4. Houck, L. D. Am. Zool. 38, 108–117 (1998).
5. Lazarus, L. H. & Attila, M. Prog. Neurobiol. 41, 473–507
(1993).
6. Erspamer, V. Amphibian Biology (ed. Heatwole, H.) 178–350
(Surrey Beatty, Chipping Norton, New South Wales, Australia,
1993).
7. Wong, H., Bowie, J. H. & Carver, J. A. Eur. J. Biochem. 247,545–557 (1997).
8. Steinborner, S. T., Currie, G. J., Bowie, J. H., Wallace, J. C. &
Tyler, M. J. J. Peptide Res. 51, 121–126 (1998).
9. Birch, M. C. in Pheromones Vol. 32 (eds Neuberger, A. & Tatum,
E. L.) 1–14 (North Holland, Amsterdam, 1974).
10.Kikuyama, S. et al. Science 267, 1643–1645 (1995).
11.Tyler, M. J., Stone, D. J. M. & Bowie, J. H. J. Pharmacol. Toxicol.
Methods 28, 199–200 (1992).
12.Wabnitz, P. A., Walter, H., Tyler, M. J., Wallace, J. C. & Bowie, J. H.
J. Peptide Res. 52, 477–483 (1998). oxygen atom. If this is the case, then DHPrepresents a new class of enzyme: adehaloperoxygenase. Its mechanism ofaction is probably similar to that of otherperoxidases, given the important role of thehigh-valency iron–oxo intermediate that isformed when hydrogen peroxide is addedto the enzyme and the subsequent het-erolytic cleavage of the O–O bond3. Theability of peroxidases to form such an inter-mediate, which globins cannot, is consistentwith the idea4 that the proximal histidineand polar residues in the distal pocket arecrucial.
Peroxidases form a strong hydrogenbond between the Nd1 nitrogen atom of theproximal histidine and a nearby glutamate,so the proximal histidine takes on a partialhistidinate character. In contrast, the proxi-mal pocket of globins lacks a glutamate, sothey form only a weak hydrogen bond4. InDHP, a hydrogen bond forms that isstronger than in globins but, rather thanbonding to a carboxylate, it is generated bya roughly 60° rotation of the imidazole ring.Its hydrogen points directly into the oxygenatom of the peptide carbonyl group of theleucine at residue 83. The hydrogen bond isshorter than that of myoglobin, in which itis bifurcated (Fig. 1). The reorientation ofthe axial ligand is facilitated by a shift of theproximal histidine in the sequence by tworesidues.
In peroxidases, the distal histidine func-tions as an acid or base in the transfer of
brief communications
NATURE | VOL 401 | 30 SEPTEMBER 1999 | www.nature.com 445
protons to the leaving water molecule, andthe developing negative charge is stabilizedby an arginine side chain4. This arrange-ment is possible because peroxidases do notuse the distal cavity as an organic-substrate-binding pocket, as the reaction takes placeat the haem edge5.
We propose that DHP binds peroxideand uses the distal histidine to cleave theO–O bond. When the high-valencyiron–oxo intermediate is ready, the distalhistidine swings out of the cavity, enablingthe substrate to enter the distal pocket andundergo oxidation. This hypothesis is sup-ported by the observation that the distalhistidine in the native DHP is disorderedbetween two positions, with one outside thedistal pocket and the other inside (Fig. 1),whereas it is outside the pocket in the com-plex with 4-iodophenol.
The distance between the Ne2 atom ofthe distal histidine and the haem iron inglobins is 4.1 to 4.6 Å, whereas in peroxi-dases it is 5.5 to 6.0 Å (ref. 6). In DHP, thisdistance is 5.4 Å for the position inside thedistal cavity, which is related to a 2.4 Å shiftof its a-carbon compared with myoglobin.The shift appears to be generated by thereplacement of the glycine that follows thedistal histidine in myoglobin by threoninein DHP.
DHP serves primarily to dehalogenatebiologically generated bromophenols, but itcan also dehalogenate trichlorophenols andother anthropogenic pollutants. The effi-ciency of the enzyme is not significantlyaffected by the position and number ofhalogen atoms at the phenol ring2. Theenzyme simply binds its organic substrateclose to a highly reactive oxygen atom andso it does not need a system involvinghydrogen bonds and other interactions thatare features of enzymes with more sophisti-cated mechanisms. This mechanism meansthat its specificity can be altered by modify-ing the residues lining the distal cavity,making it potentially useful as a tool forbioremediation projects.Lukasz Lebioda*, Michael W. LaCount*, Erli Zhang*†, Yung Pin Chen‡, Kaiping Han‡, Margaret M. Whitton†,David E. Lincoln‡, Sarah A. Woodin‡Departments of *Chemistry and ‡Biochemistry andBiological Sciences, University of South Carolina,Columbia, South Carolina 29208, USAe-mail: [email protected]†Parke-Davis Pharmaceutical Research, Division of Warner-Lambert, Ann Arbor, Michigan 48105, USA
1. Woodin, S. A., Marinelli, R. L. & Lincoln, D. E. J. Chem. Ecol.19, 517–530 (1993).
2. Chen, Y. P., Woodin, S. A., Lincoln, D. E. & Lovell, C. R. J. Biol.Chem. 271, 4609–4612 (1996).
3. Dawson, J. H. Science 240, 433–439 (1988).4. Poulos, T. L. Adv. Inorg. Biochem. 7, 1–36 (1988).5. Ator, M. A. & Ortiz de Montellano, P. R. J. Biol. Chem. 262,
1542–1551 (1987).6. Matsui, T., Ozaki, S., Liong, E., Phillips, G. N. & Watanabe, Y.
J. Biol. Chem. 274, 2838–2844 (1999).
Figure 1 The least-squares superposition of the haem groups
of dehaloperoxidase (green) and myoglobin (Protein Data Bank
entry code, 1mbo), showing the most important differences in
their environment. The position of the distal histidine of dehaloper-
oxidase out of the binding pocket is shown in cyan, and myoglobin
residues are purple.
Protein structure
An enzymatic globinfrom a marine wormSome marine worms, such as Thelepus cris-pus and Notomastus lobatus, secrete bromi-nated aromatic molecules and otherhalogenated metabolites as repellants1.Other species, such as Amphitrite ornata, donot produce repellants but are adapted tothe chemical warfare of N. lobatus andcohabit with them in estuarine mudflats.The halocompounds are tolerated by A.ornata as they are degraded by dehaloper-oxidase (DHP)2. We have determined theamino-acid sequence and crystal structureof DHP and find that its fold is typical ofthe globin family, indicating that theenzyme evolved from an oxygen carrierprotein. Residues at the dimer interface donot correspond to those in tetrameric anddimeric haemoglobins and the spatialarrangement of the dimers is different. Thecomplete amino-acid sequence is most sim-ilar to that of myoglobin from the sea hare,with 20.6% identity among 126 overlappingamino acids.
The halophenols bind in the distal pocketof DHP, indicating that DHP removes thehalogen by directly attacking an activated
