Aspartate Dehydrogenase, a Novel Enzyme Identified from Structural and Functional Studies of TM1643*

Zhiru YangDagger , Alexei Savchenko§, Alexander Yakunin, Rongguang Zhang||, Aled Edwards§, Cheryl Arrowsmith§, and Liang TongDagger **

From the Dagger  Department of Biological Sciences, Columbia University, New York, New York 10027, the § Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada, the  Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada, and the || Structural Biology Center, Argonne National Laboratory, Argonne, Illinois 60439

Received for publication, November 21, 2002, and in revised form, December 20, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

The open reading frame TM1643 of Thermotoga maritima belongs to a large family of proteins, with homologues in bacteria, archaea, and eukaryotes. TM1643 is found in an operon with two other genes that encode enzymes involved in the biosynthesis of NAD. In several bacteria, the gene in the position occupied by TM1643 encodes an aspartate oxidase (NadB), which synthesizes iminoaspartate as a substrate for NadA, the next enzyme in the pathway. The amino acid sequence of TM1643 does not share any recognizable homology with aspartate oxidase or with other proteins of known functions or structures. To help define the biological functions of TM1643, we determined its crystal structure at 2.6Å resolution and performed a series of screens for enzymatic function. The structure reveals the presence of an N-terminal Rossmann fold domain with a bound NAD+ cofactor and a C-terminal alpha +beta domain. The structural information suggests that TM1643 may be a dehydrogenase and the active site of the enzyme is located at the interface between the two domains. The enzymatic characterization of TM1643 revealed that it possesses NAD or NADP-dependent dehydrogenase activity toward L-aspartate but no aspartate oxidase activity. The product of the aspartate dehydrogenase activity is also iminoaspartate. Therefore, our studies demonstrate that two different enzymes, an oxidase and a dehydrogenase, may have evolved to catalyze the first step of NAD biosynthesis in prokaryotes. TM1643 establishes a new class of amino acid dehydrogenases.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

The open reading frame (ORF)1 TM1643 was identified in the genome of the hyperthermophilic bacterium Thermotoga maritima (1). This ORF encodes a soluble, 241-residue protein that is conserved among a large number of organisms, including Caenorhabditis elegans and humans (Fig. 1). Currently, there are more than 15 homologs of this protein in the data base, suggesting it may have an important, conserved function in these living systems. However, the function of this protein cannot be deduced from its sequence, because it does not share any recognizable similarity to other proteins of known function or structure.


View larger version (67K):
[in this window]
[in a new window]
 
Fig. 1.   Amino acid sequence conservation of TM1643. The sequence of TM1643 is aligned with its homologs in Methanoccocus jannaschii (MJ0915) and humans. A dash indicates a residue that is identical to that in TM1643, and a dot represents a deletion.

TM1643 is predicted to be the first gene in a three-gene operon in T. maritima (1). The second and third genes of this operon have sequence homology to NadA and NadC in Escherichia coli, respectively, which encode proteins that catalyze the de novo biosynthesis of NAD from L-aspartate. In prokaryotes, the first reaction in this pathway is catalyzed by the FAD-containing L-aspartate oxidase (NadB), which oxidizes L-aspartate to iminoaspartate using oxygen or fumarate as electron acceptors (2) (Fig. 2). Iminoaspartate is then condensed with dihydroxyacetone phosphate to produce quinolinate, a reaction catalyzed by NadA (quinolinate synthetase A). The third reaction in this pathway is catalyzed by NadC (quinolinate phosphoribosyltransferase). These three genes, nadB-nadA-nadC, make an operon in thermophilic bacteria (Pyrococcus, Sulfolobus) and in Bacillus. Although the TM1643 gene occupies the equivalent position of NadB in the operon, it does not share any recognizable sequence similarity to NadB.


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 2.   Reaction catalyzed by amino acid dehydrogenases. The natural function of TM1643 in vivo is likely the production of iminoaspartate, the first step of the reactions depicted, whereas that of other amino acid dehydrogenases is the oxidative deamination. The iminoaspartate product is unstable in aqueous solution and can decompose to oxaloacetate and pyruvate, as indicated by the reactions in the square brackets.

To help define the functional role of this ORF, we have determined its crystal structure at 2.6 Å resolution as part of our structural genomics effort. The structure revealed that TM1643 may be an NAD(P)+-dependent oxidoreductase. Based on the structural information, our enzymatic characterization of this protein showed that it is a dehydrogenase strictly specific for L-aspartate. Our structural and functional studies, together with the position of TM1643 in the Nad operon, therefore suggest that TM1643 and its homologues are aspartate dehydrogenases that catalyze the first step in NAD biosynthesis from aspartate. These results establish a new class of amino acid dehydrogenases. Both aspartate oxidase and aspartate dehydrogenase produce iminoaspartate from the oxidation of L-aspartate (Fig. 2). The difference between them is that the former catalyzes the reduction of FAD (and ultimately oxygen or fumarate), whereas the latter catalyzes the reduction of NAD(P).

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Expression and Purification-- Recombinant protein was expressed with a hexa-histidine fusion tag at the N terminus in E. coli BL21 (DE3). The cells were lysed in a buffer containing 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, and 0.5 mM benzamidine. The soluble recombinant protein was bound to Ni+2 affinity resin and eluted in a buffer containing 50 mM Hepes, pH 7.5, 500 mM NaCl, 5% (v/v) glycerol, and 250 mM imidazole. The purified protein was dialyzed extensively against a buffer containing 10 mM Hepes and 500 mM NaCl, concentrated to 20 mg/ml, and stored at 4 °C.

Crystallization, Data Collection, and Data Processing-- Crystals were grown at room temperature using the hanging-drop vapor diffusion method. The reservoir solution contained 0.2 M KH2PO4, 22% polyethylene glycol 1500, and 1 mM EDTA. Crystals were transferred to a cryoprotectant solution containing 25% ethylene glycol and flash-frozen in liquid nitrogen for data collection at 100 K. A single-wavelength anomalous diffraction (SAD) data set to 2.6 Å resolution was collected at the National Synchrotron Light Source beam lines X4A. The crystal belongs to space group P41212, with cell parameters of a = b = 63.2 Å and c = 125.1 Å. The diffraction images were processed with the HKL package (Table I) (3).

Structure Determination and Refinement-- The Se sites were located with the SnB program based on the anomalous differences (4). Reflection phases were calculated and solvent-flattened with the program Solve (5). The atomic model was built into the electron density map with the program O (6), and the structure refinement was carried out with the program CNS (7). The crystallographic information is summarized in Table I.

Enzymatic Studies-- Dehydrogenase activity against pools of different substrates (20 amino acids, 9 organic acids, or 8 alcohols) was measured spectrophotometrically by following the increase of absorbance at 340 nm. General assays were performed at 70 °C in a reaction mixture containing 100 mM Tricine buffer (pH 8.5), 0.5 mM NADP+, 0.5 mM NAD+, and 5 mM of the substrate mixture. Dehydrogenase activity with individual amino acids as substrates was measured in a reaction mixture containing 50 mM diethanolamine buffer (pH 9.8), 1 mM NADP+ (or NAD+), and 5 mM of the amino acid. Asp dehydrogenase activity in the direction of reductive amination (NADPH or NADH oxidation) was monitored at 70 °C as the decrease at 340 nm in a 1-ml mixture containing 50 mM Tris-HCl (pH 8.0), 100 mM (NH4)2SO4, 0.1 mM EDTA, 0.15 mM NADPH (or NADH), 5 mM oxaloacetate, and appropriate amount of enzyme.

The products of the reaction with L-aspartic acid were detected using enzymatic assays. NH4+ was measured with glutamate dehydrogenase (8). Pyruvate was measured with lactate dehydrogenase (9) and with alanine dehydrogenase (10).

The spectrophotometric assay for L-Asp oxidase activity was carried out using the horseradish peroxidase-coupled method by following the oxidation of o-dianisidine at 460 nm (11). The aspartate semialdehyde dehydrogenase (ASA-DH) activity in the biosynthetic direction (the synthesis of L-aspartate-beta -semialdehyde) was examined by using the previously described aspartate kinase-coupled method (12). The ASA-DH activity in the phosphorylating direction was assayed at 70 °C with aspartate semialdehyde as a substrate (a kind gift from Dr. Wright) by following the increase in the absorbance at 340 nm as described previously (13). The inositol-1-phosphate synthase activity was examined as previously described (14).

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Crystal Structure of TM1643-- The crystal structure of the TM1643 ORF of T. maritima in complex with NAD+ has been determined at 2.6 Å resolution by the seleno-methionyl SAD method (15). The current R factor of the model is 22.6%. The crystallographic information is summarized in Table I. The atomic coordinates have been deposited at the Protein Data Bank with the accession code 1H2H. While our structure determination was in progress, another research group deposited the structure of this protein in the PDB (entry 1J5P). The rms distance for all the equivalent Calpha atoms of the two structures is 0.3 Å.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Summary of crystallographic information

The structure of TM1643 contains two domains. The backbone fold of the N-terminal domain (residues 1-105) is essentially the same as that of the canonical Rossmann fold, with a central six-stranded parallel beta -sheet (Fig. 3A). The NAD+ molecule is associated with this domain at a position that is equivalent to the binding site in other Rossmann folds. This structural observation immediately suggests that TM1643 may be an oxidoreductase.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3.   Crystal structure of TM1643 in complex with NAD+. A, schematic drawing of the structure of TM1643 in complex with NAD+. B, schematic drawing of the dimer of TM1643. The two monomers are yellow and green, respectively. Produced with Ribbons (23).

The C-terminal domain (residues 113-241) has an open-faced sandwich structure with a five-stranded mixed beta -sheet with three helices on one side (Fig. 3A). The nicotinamide ring of NAD+ is located at the interface between the two domains, indicating that this likely is the location of the active site of this enzyme (Fig. 3A).

The C-terminal domain also helps to mediate the dimerization of the protein. The beta -sheets in the C-terminal domains of the two monomers are arranged in a side-by-side fashion, and this creates a ten-stranded beta -sheet in the dimer (Fig. 3B). In addition, the C-terminal residues of one monomer form a short beta -strand, and it extends the six-stranded beta -sheet in the N-terminal domain of the other monomer (Fig. 3B). Our structural observation suggests that TM1643 is likely to exist as dimers in solution.

Structural Conservation with Several Dehydrogenases-- Searches with the program Dali against the data base of known protein structures (16) revealed that TM1643 has weak structural homology to several other NAD(P)+-dependent oxidoreductases, including inositol 1-phosphate synthase (17), dihydrodipicolinate reductase (18), and ASA-DH (19). The amino acid sequence identity among the structurally equivalent residues is below 15%, underscoring the lack of sequence homology between TM1643 and other proteins. As could be expected, this structural homology is stronger in the N-terminal Rossmann fold domain. The core of the C-terminal beta -sheet is similar among these structures, although there are also significant variations such as the presence of additional strand(s) in the sheet. Overall, the structural homology strongly supports the notion that TM1643 is also an oxidoreductase (dehydrogenase). In addition, open and closed forms of the structures have been observed from studies on these other enzymes, with changes in the relative positioning of the two domains. Our structure of TM1643 appears to be in an open form.

The Active Site of TM1643-- The active site of TM1643 is located at the interface between the N- and C-terminal domains of the monomer. Residues in this active site come from strands beta 7 and beta 10, helices alpha A, alpha F, and alpha G, the beta 5-alpha C loop and the alpha E-alpha F loop, and finally the linker between the two domains (residues 106-112) (Fig. 4A). They are generally well conserved among this family of proteins (Fig. 1). There is a clear depression on the surface of the protein, delineating the region for substrate binding and catalysis (Fig. 4B). This active site region is open to the solvent in the current structure (Fig. 4B). It is expected that the active site will be shielded from the solvent in the closed form of the enzyme when the substrate is bound.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 4.   Active site of TM1643. A, stereo diagram showing the active site region of TM1643. B, molecular surface of TM1643 in the active site region. Panel A produced with Ribbons (23) and panel B with Grasp (24).

Our structural analysis showed that the His-193 residue may function as the general acid/base in the catalysis by these enzymes. This residue is strictly conserved among all family members and may be located near the C4 position of the nicotinamide ring in the closed form of the enzyme. In support of this observation, the His-193 residue is structurally equivalent to the catalytic His residue in ASA-DH (19).

A short loop of the enzyme, residues 212-219 (just prior to helix alpha G, Figs. 2A and 3A), is disordered in our current structure. These residues may be located in the active site, helping form a flap over the active site when the substrate is bound.

Functional Studies with TM1643-- Although TM1643 is located in the position corresponding to the aspartate oxidase gene (nadB) of the Nad operons, it does not share any sequence or structural homology with L-aspartate oxidases. Furthermore, purified TM1643 showed no evidence for the presence of an FAD cofactor, and the enzyme has no L-aspartate oxidase activity (data not shown). It is therefore highly unlikely that TM1643 is an aspartate oxidase.

TM1643 shows structural homology to ASA-DH. However, it is unlikely to be an ASA-DH because its active site lacks the Cys residue required for the catalysis by ASA-DH (19). Our enzymatic studies with purified TM1643 showed no ASA-DH activity in either the forward or the reverse reaction. In addition, purified TM1643 has no homoserine dehydrogenase or inositol-1-phosphate synthase activity, the latter enzyme being a structural homolog of TM1643.

With the information obtained from the structural studies, and from the analysis of the operon sequence conservation, we next performed enzymatic screens to explore various compounds as potential substrates for the presumed TM1643 dehydrogenase activity.

Characterization of the TM1643 Dehydrogenase-- The structural information suggested that TM1643 would likely possess dehydrogenase activity. To identify substrate(s) for TM1643, we designed general dehydrogenase screens using a mixture of NAD and NADP as the electron acceptors and three mixtures of substrates (amino acids, organic acids, or alcohols) as electron donors. Significant dehydrogenase activity was observed only with the pool of amino acids. When tested individually as electron donors, TM1643 showed robust dehydrogenase activity toward L-aspartate. No activity was found with D-aspartate; the enzyme is strictly specific for L-aspartate. L-glutamate and asparagine are not substrates of this enzyme.

Purified TM1643 showed classical Michaelis-Menten kinetics, and linear double-reciprocal plots were obtained with all three substrates tested, L-aspartate, NADP, and NAD (Fig. 5, A and B). The TM1643 kinetic parameters are presented in Table II. Interestingly, the Km for L-Asp was ~20 times lower in the presence of NAD than in the presence of NADP (Fig. 5B and Table II). Noting the lower Km for NAD and L-aspartate, it is likely that the TM1643 dehydrogenase uses NAD as the cofactor for the oxidation of aspartate in vivo. This is also consistent with our observation that NAD was co-purified and co-crystallized with TM1643 (Fig. 3A).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5.   Enzymatic studies of TM1643. A, double-reciprocal plot of the kinetic data as a function of the NADP concentration in the presence of saturating amount of aspartate. B, double-reciprocal plot as a function of the NAD+ concentration. C, catalytic activity of TM1643 as a function of the aspartate concentration in the presence of saturating concentrations of NAD+ or NADP+. Inset, details for the NAD+ curve at low concentrations of aspartate.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Steady-state kinetic parameters for TM1643 and E. coli aspartate oxidase

The L-aspartate dehydrogenase activity of TM1643 was not affected by the addition of L-glutamate, succinate, fumarate, pyruvate, tartrate, or D-malate, whereas L-malate and NH4+ were competitive inhibitors with Ki = 4.02 ± 0.48 mM and 32.5 ± 4.9 mM, respectively.

Like the reaction catalyzed by aspartate oxidase, aspartate dehydrogenase is also expected to produce iminoaspartate from the oxidation of L-aspartate (Fig. 2). In aqueous solution, however, the iminoaspartate product is not stable, and its hydrolysis will produce ammonia and oxaloacetate (Fig. 2).

We examined the products formed by the reaction of L-aspartate dehydrogenase (besides NADPH and NADH). Ammonium was detected in almost equimolar quantities to the NADH produced (data not shown). We were unable to detect oxaloacetate, however, presumably because it is very unstable under the experimental conditions that we used (pH 9.8 and 70 °C). Oxaloacetate is likely to quickly decarboxylate to pyruvate, and we were able to identify the production of pyruvate by using two enzymatic assays (data not shown). We could also demonstrate the reverse reaction, reductive amination of oxaloacetate by purified TM1643 at 70 °C and pH 8.0, using reaction conditions previously described for glutamate dehydrogenase (8). In this reverse reaction, NADPH and NADH were equally effective as electron donors.

Possible Role for TM1643 in NAD Biosynthesis-- Our structural and enzymatic studies demonstrate that TM1643 is a novel amino acid dehydrogenase that is strictly specific for L-aspartate. This raises a question concerning its functional role in T. maritima. T. maritima contains a well characterized glutamate dehydrogenase that plays a key role in linking the carbon and nitrogen metabolism in all organisms. Therefore, it is unlikely that the TM1643 aspartate dehydrogenase plays the same role in T. maritima.

TM1643 does not share direct structural similarity to the superfamily of leucine, valine, glutamate, and phenylalanine dehydrogenases (20) and thereby establishes a new class of amino acid dehydrogenases. These other enzymes catalyze the oxidative deamination of the amino acid substrates (21, 22) (Fig. 2). In contrast, TM1643 is part of the Nad operon in T. maritima, and our BLAST analysis did not reveal the presence of an L-aspartate oxidase homolog in T. maritime. We propose therefore that in this organism TM1643 catalyzes the first reaction of de novo biosynthesis of NAD from aspartate, and it produces iminoaspartate required for this pathway. The formation of an enzyme complex between TM1643 and NadA, the next enzyme of the pathway, may allow the channeling of this unstable product directly to the NadA active site.

    ACKNOWLEDGEMENTS

We thank Hailong Zhang, Reza Khayat, Xiao Tao, and Damara Gebauer for help with data collection at synchrotron radiation sources, Randy Abramowitz and Craig Ogata for setting up the X4A beamline, Ron Viola for helpful discussions regarding ASA-DH, and Gerry Wright and David Bareich for help with ASA-DH assay.

    FOOTNOTES

* This work was supported by a grant to the Northeast Structural Genomics Consortium (the Department of Biological Sciences, Columbia University, the Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, and Banting and Best Department of Medical Research, University of Toronto) from the Protein Structure Initiative of the National Institutes of Health (P50 GM62413), the Ontario Research and Development Challenge Fund, and Genome Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed. Tel.: 212-854-5203; Fax: 212-854-5207; E-mail: tong@como.bio.columbia.edu.

Published, JBC Papers in Press, December 21, 2002, DOI 10.1074/jbc.M211892200

    ABBREVIATIONS

The abbreviations used are: ORF, open reading frame; ASA-DH, aspartate semialdehyde dehydrogenase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

1. Nelson, K. E., Clayton, R. A., Gill, S. R., Gwinn, M. L., Dodson, R. J., Haft, D. H., Hickey, E. K., Peterson, J. D., Nelson, W. C., Ketchum, K. A., McDonald, L., Utterback, T. R., Malek, J. A., Linher, K. D., Garrett, M. M., Stewart, A. M., Cotton, M. D., Pratt, M. S., Phillips, C. A., Richardson, D., Heidelberg, J., Sutton, G. G., Fleischmann, R. D., Eisen, J. A., Fraser, C. M., et al.. (1999) Nature 399, 323-329[CrossRef][Medline] [Order article via Infotrieve]
2. Nasu, S., Wicks, F. D., and Gholson, R. K. (1982) J. Biol. Chem. 257, 626-632[Abstract/Free Full Text]
3. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307-326
4. Weeks, C. M., and Miller, R. (1999) J. Appl. Crystallogr. 32, 120-124[CrossRef]
5. Terwilliger, T. C., and Berendzen, J. (1999) Acta Crystallogr. Sect. D Biol. Crystallogr. 55, 849-861[CrossRef][Medline] [Order article via Infotrieve]
6. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve]
7. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve]
8. Bergmeyer, H. U. (1985) Methods Enzymol. 8, 454-461
9. Bergmeyer, H. U. (1985) Methods Enzymol. 6, 570-577
10. Yoshida, A., and Freese, E. (1970) Methods Enzymol. 17, 176-181
11. Tedeschi, G., Negri, A., Mortarino, M., Ceciliani, F., Simonic, T., Faotto, L., and Ronchi, S. (1996) Eur. J. Biochem. 239, 427-433[Abstract]
12. Angeles, T. S., and Viola, R. E. (1990) Arch. Biochem. Biophys. 283, 96-101[Medline] [Order article via Infotrieve]
13. Karsten, W. E., and Viola, R. E. (1991) Biochim. Biophys. Acta 1077, 209-219[Medline] [Order article via Infotrieve]
14. Wong, Y. H., Mauck, L. A., and Sherman, W. R. (1982) Methods Enzymol. 90, 309-314[Medline] [Order article via Infotrieve]
15. Hendrickson, W. A. (1991) Science 254, 51-58[Medline] [Order article via Infotrieve]
16. Holm, L., and Sander, C. (1993) J. Mol. Biol. 233, 123-138[CrossRef][Medline] [Order article via Infotrieve]
17. Norman, R. A., McAlister, M. S. B., Murray-Rust, J., Movahedzadeh, F., Stoker, N. G., and McDonald, N. Q. (2002) Structure 10, 393-402[CrossRef][Medline] [Order article via Infotrieve]
18. Scapin, G., Blanchard, J. S., and Sacchettini, J. C. (1995) Biochemistry 34, 3502-3512[Medline] [Order article via Infotrieve]
19. Hadfield, A., Shammas, C., Kryger, G., Ringe, D., Petsko, G. A., Ouyang, J., and Viola, R. E. (2001) Biochemistry 40, 14475-14483[CrossRef][Medline] [Order article via Infotrieve]
20. Turnbull, A. P., Baker, P. J., and Rice, D. W. (1997) J. Biol. Chem. 272, 25105-25111[Abstract/Free Full Text]
21. Rife, J. E., and Cleland, W. W. (1980) Biochemistry 19, 2328-2333[Medline] [Order article via Infotrieve]
22. Tally, J. F., Maniscalco, S. J., Saha, S. K., and Fisher, H. F. (2002) Biochemistry 41, 11284-11293[CrossRef][Medline] [Order article via Infotrieve]
23. Carson, M. (1987) J. Mol. Graphics 5, 103-106[CrossRef]
24. Nicholls, A., Sharp, K. A., and Honig, B. (1991) Proteins 11, 281-296[Medline] [Order article via Infotrieve]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.