PROTEINS: Structure, Function, and Genetics 24:407-408 (1996)

Crystallization and Preliminary Crystallographic Studies of 3-Deoxy-D-Manno-Octulosonate-8-Phosphate Synthase From Escherichia coZi William D. Tolbert, Jonathan R. Moll, Ronald Bauerle, and Robert H. Kretsinger Department of Biology, University of Virginia, Charlottesville, Virginia 22903

ABSTRACT 3-Deoxy-D-manm-octulosonate- 8-phosphate (KDOP) synthase catalyzes the production of KDOP from phosphoenolpyru- vate (PEP) and arabinose-5-phosphate (A5P). In gram-negative bacteria KDOP is subse- quently dephosphorylated, cytidylylated, and linked to lipid A and is required for lipid A in- corporation into the outer membrane (Raetz, Annu. Rev. Biochem. 59129-170,1990). We have crystallized two forms of KDOP synthase be- longing to space groups I23 or I2,3, one with a = b = c = 118.0 A and the other with a = b = c = 233 A. 0 1996 Wiley-Liss, Inc.

Key words: KDOP synthase, lipopolysaccha- ride biosynthesis, X-ray crystallog- raphy

MATERIALS AND METHODS Expression and Purification

The kdsA expression vector pCAM5 was con- structed by the cloning of the kdsA, which encodes KDOP synthase, from plasmid pMWlOl into plas- mid pttG1.l Plasmid pttGl is an expression vector for the aroG gene under the control of tandem tac and aroG promoters, the aroG Shine-Dalgarno se- quence, and the rpoC terminator.' NdeI sites (5' CATATG 3') were created by oligonucleotide site- directed mutagenesis a t the beginning of both the kdsA and aroG coding sequences. The entire aroG gene of pttGl was then excised as an NdeI-HinDIII fragment and replaced with the NdeI-HinDIII frag- ment from the pMWlOl derivative containing the entire kdsA gene. Nutritional tests showed that pCAM5 complements a Salmonella typhimurium kdsAt" mutant at non-permissive temperatures.,

KDOP synthase was purified from cultures of E . coli strain CB198 (W3110 trpR tnaA lacIq) carrying pCAM5. Cells were grown in a New Brunswick fer- mentor in 10 L M9 minimal media supplemented with 0.1% casamino acids.4 Induction of KDOP syn- thase was carried out by the addition of 0.5 mM isopropyl-P-D-thiogalactopyranoside when the cul- ture reached an Abs,,, of 0.3. When the Abs,,, reached 2.3, the cells were collected by centrifuga- tion and resuspended in buffer A [ZO mM bis-tris propane (BTP), pH 6.25, and 1.0 mM dithiothreitol

1996 WILEY-LISS, INC.

(DTE)]. The cells were then sonicated and centri- fuged. Ammonium sulfate was added to the crude supernatant to a concentration of 55% saturation and the precipitate was collected by centrifugation at 17,500g for 20 minutes. The pellet was discarded and ammonium sulfate was added to the superna- tant to a concentration of 70% saturation. The prep- aration was centrifuged at 17,500g for 30 minutes and the pellet resuspended in 20 ml of buffer A and dialyzed against buffer A with three changes of di- alysis buffer over 24 hours. Dialyzed protein was then applied to a 1.0 L Amicon Matrex Gel Orange A column. The column was washed with three volumes of buffer A. KDOP synthase was then eluted with buffer A containing 400 pM PEP. PEP was removed by dialysis against 50 mM 3-[N-morpholinolpro- panesulfonic acid (MOPS), pH 7.0, and 1.0 mM DTE with three changes of dialysis buffer over 24 hours. KDOP synthase accounted for approximately 15% of the total protein in crude extracts. Ninety milli- grams of pure KDOP synthase can be isolated per liter of culture.

Enzymatic Assay KDOP synthase activity was determined by the

rate of disappearance of PEP measured in a record- ing spectrophotometer at OD,,, using a molar ex- tinction coefficient of 2,790 M-lcm-' for PEP. The standard reaction mixture contained 100 mM BTP, pH 7.0, 200 pM PEP, and 300 pM A5P in a final volume of 1.0 ml. One unit of activity is defined as the disappearance of 1.0 pmol PEP/mg protein a t 25°C. The concentration of KDOP synthase, ex- pressed in terms of the enzyme monomer, was de- termined spectrophotometrically using Ego = 6,650 M-lcm-' (determined gravimetrically) or enzymat- ically using a specific activity of 7.8 unitdmg.

Crystallization The purified protein was crystallized by the hang-

ing drop method. Ten microliters of 17 mg/ml of KDOP synthase and 10 pl of the well solution were

Received September 28,1995; accepted September 29,1995. Address reprint requests to Robert H. Kretsinger, Depart-

ment of Biology, Gilmer Hall, University of Virginia, Char- lottesville, VA 22903.

408 W.D. TOLBERT ET AL.

mixed and placed on a coverslip. The coverslip was then inverted and placed over the solution in one of the 24 wells of a Linbro tissue culture plate. Wells were sealed with mineral oil.

Crystals were mounted in 1.0-1.5 mm diameter quartz capillaries containing small volumes of well solution and sealed with wax. Mounted crystals were initially characterized by precession photo- graphs using Ni filtered Cu K, radiation (A = 1.54 A). Diffraction data were measured at our Multiwire Area X-ray Diffractometer f a ~ i l i t y . ~ The data acqui- sition and data reduction was carried out by a mi- croVAX 3600 computer. Subsequent scaling and data evaluation were performed with the CCP4 suite of crystallographic programs.6

RESULTS AND DISCUSSION Two crystal forms suitable for crystallographic

analysis were found: form a grown in polyethylene glycol and form p grown in ammonium sulfate. Well solutions for crystal form a varied from 20 to 28% PEG 1500,50 mM MOPS, pH 7.0,lO mM DTE, and 1.0 mM A5P. Well solutions for crystal form p varied from 45 to 53% ammonium sulfate, 50 mM potas- sium phosphate, pH 7.0, and 10 mM DTE. Crystals usually appeared within 1 or 2 weeks of incubation at 18°C. Crystal form p diffracted for approximately 3 days and crystal form a approximately 5 days when cooled to 4°C during data collection.

Precession photographs of form a show a unit cell witha = b = c = 118.0Aanda = p = y = 90”.The systematic absence of h + k + 1 = odd and the precession photographs initially indicated that the space group is I23 or I2,3. Subsequent data collec- tion and scaling have confirmed this assignment. Area detector data ranged from 41.7 to 2.8 A (Table I). Space groups I23 and I2,3 contain 24 asymmetric unitdunit cell; therefore with one monomer/asym- metric unit the crystal contains approximately 43% solvent (calculated using a protein density of 1.3 g/ml) and a V,,, = 2.22 A3/Da.7

Crystal form p was analyzed from data measured on the area detector from 165 to 4.4 A, the limit of resolution (Table I). Data were indexed with a = b = c = 233 A and a = p = y = 90”. Crystals display a systematic absence of h + k + I = odd and belong to space group I23 or I2,3. Assuming a V, of ap- proximately 2.2 as in crystal form a, 2 = 8 and the unit cell contains 192 (24 x 8) molecules with a solvent content of approximately 42%.

The smaller unit cell and better diffraction of crys- tal form a make it more amenable to structure de- termination. We are currently searching for heavy metal derivatives of form a to use in structure de- termination.

KDOP synthase has sequence identity ranging from 16 to 18% and a mechanistic similarity with the three isoforms of 3-deoxy-D-arabino-heptu-

TABLE I. Data Collection Statistics for Crystal Form Q From 41.7 A to 2.8 A and Crystal Form f3

From 165 to 4.4 A Data ranee Form a Form B 41.7-4.4A

Lattice points 1,805 Total reflections 22,074, Unique reflections 1,805 Completeness (%) 99 Redundancy 12.2 <nu> 18.0 Rmerg 3.8

Lattice points 2,266 Total reflections 14,044 Unique reflection 2,266

Redundancy 6.2 <Nu> 10.9

4.4-3.31

Completeness (%) 99

&erg (%) 7.3 3.3 -2.81

Lattice points 2,799 Total reflections 8,026 Unique reflections 1,906 Completeness (%) 68 Redundancy 4.2 <nu> 5.3 RmerEe (%) 14.4

losonate-7-phosphate (DAHP) synthase from E. coli. It is important to understand whether these simi- larities reflect divergent or convergent evolution. Our concurrent studies of the crystal structure of the Phe regulated DAHP synthase from E. coli should help resolve this question.

ACKNOWLEDGMENTS We gratefully acknowledge support from the Jef-

fress Memorial Trust to R.H.K. and the NIH (GM35889) to R.B.

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5. Sobottka, S.E., Chandross, R.J., Cornick, G.G., Kretsinger, R.H., Rains, R.G. Design and performance of the multiwire area diffractometer at the University of Virginia. J. Appl. Crystallogr. 23:199-208, 1990.

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