©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Escherichia coli Malonyl-CoA:Acyl Carrier Protein Transacylase at 1.5- Resolution
CRYSTAL STRUCTURE OF A FATTY ACID SYNTHASE COMPONENT (*)

Laurence Serre (1)(§), Elizabeth C. Verbree (2), Zbigniew Dauter (3), Antoine R. Stuitje (2), Zygmunt S. Derewenda (1)(¶)

From the (1) Medical Research Council Group in Protein Structure and Function, Department of Biochemistry, Edmonton, Alberta T6G 2H7, Canada, (2) Department of Genetics, Institute for Molecular Biological Sciences, BioCentrum Amsterdam, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands, and (3) EMBL Outstation, 22603 Hamburg, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Endogenous fatty acids are synthesized in all organisms in a pathway catalyzed by the fatty acid synthase complex. In bacteria, where the fatty acids are used primarily for incorporation into components of cell membranes, fatty acid synthase is made up of several independent cytoplasmic enzymes, each catalyzing one specific reaction. The initiation of the elongation step, which extends the length of the growing acyl chain by two carbons, requires the transfer of the malonyl moiety from malonyl-CoA onto the acyl carrier protein. We report here the crystal structure (refined at 1.5-Å resolution to an R factor of 0.19) of the malonyl-CoA specific transferase from Escherichia coli. The protein has an / type architecture, but its fold is unique. The active site inferred from the location of the catalytic Ser-92 contains a typical nucleophilic elbow as observed in / hydrolases. Serine 92 is hydrogen bonded to His-201 in a fashion similar to various serine hydrolases. However, instead of a carboxyl acid typically found in catalytic triads, the main chain carbonyl of Gln-250 serves as a hydrogen bond acceptor in an interaction with His-201. Two other residues, Arg-117 and Glu-11, are also located in the active site, although their function is not clear.


INTRODUCTION

Endogenous synthesis of fatty acids is essential to all living organisms. The reactions involved in the process are complex but can be visualized in a simplified form as an iterative process of linear decarboxylative condensations of several (normally seven) molecules of malonate onto an acetyl primer. Subsequent to condensation, each such elongation step requires NADPH-dependent modifications including ketoreduction, dehydration, and enoyl reduction (1, 3) . Individual reactions are catalyzed by the components of the fatty acid synthase (FAS)() complex. Type I FAS, found in animals and man, is a single polypeptide chain, approximately 2500 amino acids in length, and is made up of eight distinct domains including seven with discrete catalytic functions (1) . The molecule functions as a homodimer (2) . In contrast, bacteria and plants contain type II FAS, which is composed of structurally independent proteins (3) . Their exact number is species-dependent, and there may be several isoforms of some components. The fatty acid biosynthesis pathway is not unique in the way it utilizes short chain acyl-CoA thioesters as building blocks for larger organic molecules. It has been shown that analogous pathways are involved in the generation of polyketides, where multifunctional and/or multienzyme polyketide synthases catalyze the syntheses of such diverse biologically active molecules as immunosuppressants, anti-tumor drugs, and antibiotics (4, 5, 6) .

The initiation of each elongation step in the fatty acid synthesis cycle requires the transfer of a malonyl moiety from the respective CoA thioester to the -SH group of the phosphopantetheine arm of the acyl carrier protein (ACP), the central component of any FAS. Depending on the FAS type this reaction may either be catalyzed by a general transferase that equilibrates between short chain CoA and ACP thioesters (FAS I) or a specialized enzyme that is involved only in the elongation step (FAS II). The transferase domain of the rat liver FAS I has been recently characterized both at the protein and DNA levels (7, 8) . In Escherichia coli FAS II includes a specific malonyl-CoA:ACP transacylase (MCAT), a 32-kDa single chain enzyme that has been purified to homogeneity and characterized at the protein level (9, 10). More recently the fabD gene coding for this protein has been cloned and overexpressed (11) . We now report the crystal structure of native MCAT, solved by multiple isomorphous replacement and refined at 1.5-Å resolution. Apart from the two-dimensional NMR study of the E. coli ACP (12) , this report constitutes the first structural study of a component of an FAS complex.


MATERIALS AND METHODS

The enzyme was overexpressed, purified, and crystallized as described previously (13) ; only the hexagonal crystal form was used in the present study (space group P6, a = 68.2 Å, c = 118.6 Å). A preliminary search for heavy atom derivatives was carried out using the X1000 Siemens area detector mounted on a Siemens rotating anode source ( = 1.54178 Å). Three heavy atom derivatives (mercury, platinum, and gold) were identified. To improve the quality of x-ray data and to enhance the anomalous scattering effects, the final experiments were conducted using synchrotron radiation (beamlines BW7B and X31, EMBL Outstation, Hamburg, Germany) using MAResearch imaging plate systems (for further details see ). Scaling between the data sets, difference Patterson maps and other calculations were carried out using the CCP4 suite of programs (14) . The major site in the mercury derivative was identified in both isomorphous and anomalous difference Patterson maps; SIROAS (single isomorphous replacement optimized anomalous scattering) (15) phases were calculated for data between 15 and 2.4 Å using MLPHARE (16) . These were used to calculate Fourier difference maps and identify both the secondary mercury sites and the heavy atoms sites in the other two derivatives. Final phase calculation (MIROAS (multiple isomorphous replacement optimized anomalous scattering)) using all three derivatives resulted in a figure of merit of 0.64. Density modification involving solvent flattening and histogram matching using SQUASH (17) led to an average shift in phases of 20.2°. To resolve space group ambiguity phases were calculated in both P6 and P6; the latter yielded an easily interpretable map showing elements of secondary structure folded in agreement with accepted principles of protein chemistry. Model building was done using O (18) on a SGI Indigo Extreme. The initial conventional crystallographic R factor ( F - F / F ), for the model that included all amino acids from residue 4 to 306, was 0.47 for all the data between 8.0- and 2.0-Å resolution and decreased to 0.24 after one cycle of crystallographic refinement with simulated annealing using X-PLOR (19) . The resulting electron density map allowed for manual reconstruction of some loops, addition of residues 3 and 307, as well as water molecules identified using an automated procedure of ARP (20) . Further crystallographic refinement using the energy-restrained algorithm of X-PLOR resulted in an R factor of 0.17 (all data in the 8.0-2.0-Å range). Final details of the 1.5-Å refinement currently in progress (R factor of 0.19 for all the data) will be described elsewhere.


RESULTS

The Quality of the Model

The final model contains residues from 3 to 307 and 170 water molecules. The stereochemistry was assessed using PROCHECK (21) ; root mean square deviations from ideal bond lengths and angles were 0.012 Å and 2.58°, respectively. Of all amino acids, 95.4% are in the most favored regions of the Ramachandran plot. The only outlier is the active site serine 92, which is in a strained conformation ( = 50°, = -99°), characteristic of the nucleophile in / hydrolases (22, 23) . There is one cis-peptide that precedes Pro-52. The mean isotropic atomic displacement (B) factor for all atoms is 19.6 Å.

Overview of the Structure

The tertiary fold of the enzyme is best described in terms of two subdomains (Fig. 1a). The larger subdomain is made up of two non-contiguous segments including residues 3-123 and 206-307. It contains a short four-stranded parallel -sheet and 12 helices from 4 to 17 residues in length. The smaller subdomain, residues 124-205, contains a four-stranded anti-parallel -sheet capped by two -helices. The fold is, in general terms, of the / type, but (to our knowledge) is unique among known proteins.


Figure 1: A, schematic representation of the three-dimensional structure of E. coli MCAT. -Strands and -helices were defined according to the DSSP algorithm (24); helices: H1, 20-25; H2, 28-40; H3, 44-50; H5, 53-56; H6, 59-79, H7, 94-101; H8, 107-123; H9, 140-150; H10, 173-185; H11, 206-217; H12, 240-250; H13, 257-266; H14, 280-288; H15, 300-306; -strands: B1, 5-8; B2, 87-90; B3, 130-136; B4, 156-163; B5, 166-172; B6, 190-193; B7, 271-274; B8, 293-296. The major subdomain is made up of two non-contiguous parts: one containing helices H1-H8 and strands B1-B2 (yellow), and the other made up of H11-H15 and strands B7-B8 (orange). The smaller subdomain (green) is formed by a contiguous stretch including stands B3-B6 and helices H9-H10. Three amino acids forming the modified triad at the active site are shown in violet. B, stereochemistry at the active site of MCAT. The green color represents invariant residues among MCAT homologues; Ser-200 and Gln-63, both of which are mutated to Phe in FAS I, are shown in pink. Water molecules are shown in red. C, comparison of MCAT (violet) with R. miehei lipase complexed with n-hexylphosphonate ethyl ester (paleblue). H-bonds are indicated by dashedlines. The two water molecules in the active site of MCAT believed to occupy potential oxyanion binding sites are shown in orange. The yellow phosphorus-bonded oxygen shown in yellow indicates the location of the oxyanion hole in lipase. The figure was generated after C atoms of residues 90-94 of MCAT were superimposed on the corresponding atoms in residues 142-146 in the inhibited lipase. The figure was generated using RIBBONS (38).



The Active Site

The active site of MCAT is located in a gorge between the two subdomains (Fig. 1a). The nucleophile, Ser-92, is located in a sharp turn between a -strand and an -helix within the major subdomain. There is an H-bond between the side chain hydroxyl of Ser-92 and N-2 of His-201, reminiscent of similar interactions in serine hydrolases containing so-called catalytic triads (Fig. 1b). However, the N-1 of His-201 acts as a donor in an H-bond with a main chain carbonyl oxygen of Gln-250 and not a carboxyl acid typically observed in triads. The hydroxyl of Ser-92 is also within hydrogen bonding distance (3.07 Å) of N-2 of Arg-117, a completely buried residue as assessed by DSSP (24). Two well resolved water molecules are found in the proximity of Ser-92 (Fig. 1, b and c). Water 321 (B = 14 Å) accepts H-bonds from the main chain amides of Leu-93 (3.05 Å) and Gln-11 (2.87 Å) and donates an H-bond to water 335 (2.77 Å). The latter (B = 20 Å) accepts a second H-bond from N-1 of Arg-117 (3.07 Å) and donates to the hydroxyl of the Ser-92 (3.08 Å) as well as O-1 of Gln-11 (3.13 Å). Residues Ser-92, His-201, Gln-11, and Arg-117 constitute 4 out of 8 invariant residues in the hitherto sequenced homologues of MCAT ().


DISCUSSION

Early characterization of the E. coli MCAT at the protein level (9, 10) indicated that the enzyme is serine-dependent, with the catalytic residue located at the center of a GHSLG pentapeptide that compares well with the frequently invoked GXSXG consensus sequence (25) of various acylhydrolases. All hitherto determined structures of these enzymes exhibit the / hydrolase fold (23) in which the consensus pentapeptide forms a tight turn between a -strand and an -helix, a motif known as the nucleophilic elbow (23) or the - Ser- motif (22) . Furthermore, in all serine-dependent / hydrolases the active sites contain Ser-His-Asp(Glu) catalytic triads analogous with that originally described in chymotrypsin (26) . It is noteworthy that the sequence of the active site pentapeptide in MCAT is identical to the one found in a well studied family of extracellular lipases from filamentous fungi (27) and also found in the mammalian pancreatic lipases (28) . Hence, we expected that MCAT would exhibit a typical / hydrolase tertiary structure. Surprisingly, MCAT has a unique fold, albeit the 18-residue-long oligopeptide containing the active serine is structurally very close to the nucleophilic elbow of / hydrolases.() This is the first observation of the nucleophilic elbow outside the context of the / hydrolase fold and a dramatic example of convergent evolution.

The presence of the Ser-His dyad stabilized by an H-bond between the imidazole of His-201 and a main chain carbonyl group was also a surprise. An analogous stereochemistry has been recently found in an unrelated esterase from Streptomyces scabies(29) . A reassessment of the mechanism of serine hydrolases based on the comparison of MCAT with other enzymes appears elsewhere.()

It can be assumed that in MCAT the acylation step and subsequent transfer of the acyl moiety proceed via tetrahedral intermediates in a fashion similar to the hydrolytic reaction pathway of serine proteinases. There is, however, a major difference between MCAT and the hitherto characterized hydrolases: the acyl-enzyme complex of MCAT is stable in aqueous solution, and deacylation can only take place in the presence of specific thiol acceptors.() The comparison of the active site of MCAT with that of the Rhizomucor miehei lipase complexed with n-hexylphosphonate ethyl ester (entry 5TGL in the Protein Data Bank, Brookhaven National Laboratory; Ref. 32), a transition state analogue, provides some clues as to the possible structural roots of this difference. There are two possible sites for the oxyanion hole in MCAT occupied by solvent molecules water 335 and water 321, respectively (see ``Results,'' Fig. 1c). However, with the exception of the main chain NH of Leu-93 the potential H-bond donors (NH of Gln-11; N-1 of Arg-117, and N-2 of Gln-11() ) are located 1.0-1.5 Å further away from the nucleophile than in other serine hydrolases. It is this relative displacement of the putative oxyanion hole from the nucleophile in MCAT that may be responsible for the absence of hydrolytic activity. Assuming that a potentially hydrolytic water molecule would approach the carbonyl carbon of the acyl-enzyme adduct in a fashion similar to that seen in serine proteinases (33, 34) , there are no interfering steric constraints in the MCAT structure. However, if the target carbon is pulled away from His-201 into the oxyanion hole, the water molecule cannot be suitably oriented and activated by an H-bond to N-2 of His-201. Since sulfur has a larger radius than oxygen and a C-S bond is 0.4 Å longer than a C-O bond, -SH might be more suitable as an acceptor, as indeed is the case in MCAT.

Finally, we address the critical issue of substrate specificity. In FAS I the transferase domain is capable of equilibrating various short chain acyl-CoA thioesters with respective acyl-ACP complexes. This includes acetyl-CoA and malonyl-CoA, both of which are involved in the fatty acid synthesis process (1, 3) , as well as methylmalonyl-CoA in the biosynthesis of some methyl branched fatty acids, and propionyl-CoA in the priming reaction leading to the synthesis of fatty acids with an odd number of carbon atoms. The synthetic route is in this case determined by the available pool of CoA thioesters (35) . The bacterial MCAT, on the other hand, is highly specific toward malonyl-CoA and does not participate in the priming transfer of the acetyl moiety onto ACP (3) . In the polyketide synthase, the analogues of MCAT most commonly use methylmalonyl-CoA (36) . Thus, these pathways are determined by the transferase specificity.

The structure of the native E. coli MCAT fails to provide an obvious rationale for its specificity. It is tempting to speculate the Arg-117 might play a role in the binding of the free carboxyl group as is the case, for example, in citrate synthase (37) . This arginine, however, is invariant among all known MCAT homologues (), including two sequenced FAS I transferase domains that shuttle the acetyl group with equal ease. We note that in these two cases there are non-conservative mutations within the substrate binding site, replacing polar Ser-200 and Gln-63 (MCAT notation) with hydrophobic phenylalanines (Fig. 1b). Mutational studies, under way in our laboratories, will no doubt shed more light on the structure-function relationships in this interesting family of enzymes.

  
Table: Data collection and phasing

Native data were collected using = 0.87 Å while derivative data were collected using = 0.993 Å (L absorption edges for mercury, gold, and platinum are 1.0091, 1.0400, and 1.0723 Å, respectively). Data were processed and reduced with the DENZO-SCALEPACK programs (39). The derivatives were prepared shortly before data collection by soaking native crystals as follows: mercury, 0.6 mM thimerosal, 12 h; platinum, 0.5 mM KPtCl for 4 days; gold, 5 mM NaAuCN for 1 day. In contrast to the original mother liquor (45-50% saturated ammonium sulfate, 0.1 M Mes, pH 6.0, 1% (w/v) -octyl glucoside) the soaking solutions were detergent-free. R is the measure of the internal consistency of the data, defined as I- I/I, where I is the intensity of the ith observation and I is the mean intensity of the reflection; R is the measure of the mean relative isomorphous difference between the native protein F and the derivative F data defined as F- F/F; phasing power is defined as F/, where F is the mean heavy atom contribution and the mean lack of closure; R is defined as /F- F.


  
Table: Partial sequence alignment of MCAT homologues: active site residues



FOOTNOTES

*
This research was supported by the Medical Research Council of Canada, by a grant to the Group in Protein Structure and Function (to Z. S. D.), by the Alberta Heart and Stroke Foundation (to Z. S. D.), and by Grant VBI 80.1412 of the Dutch Stichting voor de Technische Wetenschappen (to A. R. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomiccoordinates of MCAT (accession code 1MLA) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

§
Recipient of a postdoctoral fellowship from the Alberta Heritage Foundation for Medical Research.

Medical Scholar of the Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed: MRC Group in Protein Structure and Function, Dept. of Biochemistry, University of Alberta, 4-74 Medical Sciences Bldg., Edmonton, Alberta T6G 2H7, Canada. Tel.: 403-492-2136; Fax: 403-492-0886. E-mail: zygmunt@hal.biochem.ualberta.ca.

The abbreviations used are: FAS, fatty acid synthase; ACP, acyl carrier protein; MCAT, malonyl-CoA:ACP transacylase; Mes, 2-(N-morpholino)ethane sulfonic acid.

The root mean square difference between the coordinates of the C atoms of residues 86-103 of MCAT superposed by least squares on the analogous fragment of the Rhizomucor miehei lipase (residues 138-155, entry 3TGL in Protein Data Bank, Brookhaven National Laboratory) is 0.73 Å.

Z. S. Derewenda, Y. Wei, and L. Serre, submitted for publication.

The compounds that can serve as acceptors include CoA, pantetheine, N-(N-acetyl--alanyl)-cysteamine, and N-acetylcysteamine (31).

N-2 of Gln-11 can serve as an H-bond donor in the putative oxyanion hole only after a rotation of the side chain 180° around with respect to the structure described here.


ACKNOWLEDGEMENTS

We acknowledge Drs. Masao Fujinaga, Peter Sheffield, and Stuart Smith for discussions and helpful suggestions in the course of the preparation of the manuscript.


REFERENCES
  1. Smith, S.(1994) FASEB J. 8, 1248-1259 [Abstract/Free Full Text]
  2. Smith, S., Stern, A., Randhawa, Z. I., and Knudsen, J.(1985) Eur. J. Biochem. 152, 547-555 [Abstract]
  3. Magnuson, K., Jackowski, S., Rock, C. O., and Cronan, J. E., Jr.(1993) Microbiol. Rev. 57, 522-542 [Abstract]
  4. Donadio, S., Staver, M. J., McAlpine, J. B., Swanson, S. J., and Katz, L.(1991) Science 252, 675-679 [Medline] [Order article via Infotrieve]
  5. Shen, B., and Hutchinson, R.(1994) Science 262, 1535-1540
  6. Marshen, A. F. A., Caffrey, P., Aparicio, J. F., Loughran, M. S., Staunton, P., and Leadlay, P. F.(1994) Science 263, 378-379 [Medline] [Order article via Infotrieve]
  7. Rangan, V. S., Witkowski, A., and Smith, S.(1991) J. Biol. Chem. 266, 19180-19185 [Abstract/Free Full Text]
  8. Joshi, A., and Smith, S.(1993) Biochem. J. 296, 143-149 [Medline] [Order article via Infotrieve]
  9. Williamson, I. P., and Wakil, S. J.(1966) J. Biol. Chem. 241, 2326-2332 [Abstract/Free Full Text]
  10. Ruch, F. E., and Vagelos, P. R.(1973) J. Biol. Chem. 248, 8080-8094
  11. Verwoert, I. I. G. S., Verbree, E. C., van der Linden, K. H., Nijkamp, H. J. J., and Stuitje, A. R.(1992) J. Bacteriol. 174, 2851-2857 [Abstract]
  12. Holak, T. A., Kearsley, S. K., Kim, Y., and Prestegaard, J. H.(1988) Biochemistry 27, 6135-6142 [Medline] [Order article via Infotrieve]
  13. Serre, L., Swenson, L., Green, R., Wei, Y., Verwoert, I. I. G. S., Verbree, E., Stuitje, A. R., and Derewenda, Z. S.(1994) J. Mol. Biol. 242, 99-102 [CrossRef][Medline] [Order article via Infotrieve]
  14. CCP4 Suite of Programs for Protein Crystallography(1988) SERC, Daresbury Laboratory, Warrington, UK
  15. Baker, P. J., Farrants, G. W., Stillman, T. J., Britton, K. L., Helliwell, J. R., and Rice, D. W.(1990) Acta Crystallogr. A46, 721-725 [CrossRef]
  16. Otwinowski, Z.(1991) in Isomorphous Replacement and Anomalous Scattering, pp. 80-86, SERC, Daresbury Laboratory, Warrington, UK
  17. Zhang, K. Y. J.(1993) Acta Crystallogr. D49, 213-222 [CrossRef]
  18. Jones, T. A., Zou, J.-Y., Cowan, S. W., and Kjeldgaard, M.(1991) Acta Crystallogr. A97, 110-119
  19. Brunger, A. T.(1993) X-PLOR Manual, version 3.0, Yale University, New Haven, CT
  20. Lamzin, V. S., and Wilson, K. S.(1990) Acta Crystallogr. D49, 129-147
  21. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1992) PROCHECK Version2: Programs to Check the Stereochemical Quality of Protein Structures, Oxford Molecular Ltd., Oxford, Great Britain
  22. Derewenda, Z. S., and Derewenda, U.(1991) Biochem. Cell Biol. 69,842-851 [Medline] [Order article via Infotrieve]
  23. Ollis, D. L., Cheah, E., Cygler, M., Johnson, L. N., Egmond, M. R., and Frenken, L. G.(1992) Protein Eng. 5, 197-211 [Abstract]
  24. Kabsch, W., and Sander, C.(1983) Biopolymers 22, 2577-2637 [Medline] [Order article via Infotrieve]
  25. Brenner, S.(1988) Nature 334, 528-530 [CrossRef][Medline] [Order article via Infotrieve]
  26. Blow, D. M., Birkoft, J. J., and Hartley, B. S.(1969) Nature 221, 337-340 [Medline] [Order article via Infotrieve]
  27. Derewenda, U., Swenson, L., Green, R., Wei, Y., Dodson, G. G., Yamaguchi, S., Haas, M. J., and Derewenda, Z. S.(1994) Nature Struct. Biol. 1, 36-47 [Medline] [Order article via Infotrieve]
  28. Bourne, Y., Martinez, C., Kerfelec, B., Lombardo, D., Chapus, C., and Cambillau, C.(1994) J. Mol. Biol. 238, 709-732 [CrossRef][Medline] [Order article via Infotrieve]
  29. Wei, Y., Shottel, J. L., Derewenda, U., Swenson, L., Patkar, S., and Derewenda, Z. S.(1995) Nature Struct. Biol. 2, 218-223 [Medline] [Order article via Infotrieve]
  30. Petrovics, G., Putnoky, P., Reuhs, B., Kim, J., Thorp, T. A., Noel, K. D., Carlson, R. W., and Kondorosi, A.(1993) Mol. Microbiol. 8, 1083-1094 [Medline] [Order article via Infotrieve]
  31. Joshi, V. C., and Wakil, S. J.(1971) Arch. Biochem. Biophys. 143, 493-505 [Medline] [Order article via Infotrieve]
  32. Brzozowski, A. M., Derewenda, U., Derewenda, Z. S., Dodson, G. G., Lawson, D. L., Turkenburg, J. P., Bjorkling, F., Huge-Jensen, B., Patkar, S. A., and Thim, L.(1991) Nature 351, 491-494 [CrossRef][Medline] [Order article via Infotrieve]
  33. Dixon, M. M., and Matthews, B. W.(1989) Biochemistry 28, 7034-7037
  34. Blanchard, H., and James, M. N. G.(1994) J. Mol. Biol. 241, 574-587 [CrossRef][Medline] [Order article via Infotrieve]
  35. Buckner, J. S., Kolattukudy, P. E., and Rogers, L.(1978) Arch. Biochem. Biophys. 186, 152-163 [Medline] [Order article via Infotrieve]
  36. Katz, L., and Donadio, S.(1993) Annu. Rev. Microbiol. 47, 875-912 [CrossRef][Medline] [Order article via Infotrieve]
  37. Karpusas, M., Holland, D., and Remington, S. J.(1991) Biochemistry 30, 6029-6031
  38. Carson, M.(1987) J. Mol. Graphics 5, 103-106 [CrossRef]
  39. Otwinowski, Z.(1993) in Data Collection and Processing (Saywer, L., Isaacs, N., and Bailey, S., eds) pp. 56-62, SERC, Daresbury Laboratory, Warrington, UK
  40. Amy, C. M., Witkowski, A., Naggert, J., Williams, B., Randhawa, Z., and Smith, S.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3114-3118 [Abstract]
  41. Holzer, K. P., Liu, W., and Hammes, G. G.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4387-4391 [Abstract]
  42. Mathur, M., and Kolattukudy, P. E.(1992) J. Biol. Chem. 267, 19388-19395 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.