PDFlib PLOP: PDF Linearization, Optimization, Protection

Page inserted by evaluation versionwww.pdflib.com – sales@pdflib.com

Converting T4 Phage Lysozymeinto a Transglycosidase

RYOTA KUROKI,a KEIICHI MORIMOTO,b AND BRIAN W. MATTHEWSc

aCentral Laboratories for Key TechnologyKirin Brewery CompanyYokohama 236, Japan

bResearch and Development DivisionKirin Brewery Company

Tokyo 150, Japan

cInstitute of Molecular BiologyHoward Hughes Medical Institute

andDepartment of PhysicsUniversity of Oregon

Eugene, Oregon 97403

INTRODUCTION

Transglycosylation is a useful reaction often seen in saccharide synthesis in bio-logical systems. The elucidation of the reaction mechanism of transglycosylationmay enable us to utilize this reaction to synthesize complex molecules. There are atleast two types of glycosidases: those that have transglycosylation activity and thosethat do not. The mechanistic differences between these two types of enzymes are notwell understood. The structural information on lysozymes, one of the most investi-gated glycosidases,1–3 suggests that only small changes in the catalytic residues areenough to differentiate functional glycosidases. While the glutamic acid located onthe �-side of the saccharide is structurally almost conserved among many �-glycosi-dases,4 many variations seem to be allowed on the �-side of the saccharide.5–7 The�-side residue may therefore be a key residue in determining differences in themechanisms of these enzymes. Recently, we have succeeded in changing the catalyt-ic mechanism of T4 phage lysozyme (T4L) from a single displacement to a doubledisplacement mechanism by mutating Thr-26 to Glu or His.8,9 Further analysisshown here revealed that the T26H mutant had also acquired transglycosylation ac-tivity.

MATERIALS AND METHODS

Materials

Substitutions of Thr-26 of T4 lysozyme with Glu, Gln, Asp, and His were con-structed and purified as reported previously.10 The substrate used to analyze the cat-

362

alytic activity for the mutant lysozymes was the dimer of the peptidic tetrasaccharide,NAG-NAM-DAla-DGlu-DAPA, in which the reducing end was reduced (see FIGURE

1). This substrate is produced by the partial hydrolysis of Escherichia coli cell wallsas described.9

Analytical Methods

The reaction mixture was analyzed by HPLC with a reversed-phase column (3.9 ×150 mm) of �-Bondasphere (C18, 300 Å). The column was eluted with a gradientfrom 1% CH3CN containing 0.05% TFA to 15% CH3CN containing 0.02% TFA at aflow rate of 1.0 mL/min.

The time course of the transglycosylation reaction was investigated by the samesystem. Approximately 0.1 mM saccharide substrate with peptide was digested by 10�M mutant lysozyme in 50 mM sodium acetate buffer at pH 5.0 and 0 °C.

The molecular weight of each peak eluted from the reversed-phase column wasdetermined by a Sciex III mass spectrometer.

RESULTS AND DISCUSSION

It has already been reported that the catalytic reaction of the T26H mutant of T4Lproceeds through a covalent intermediate and is a retaining glycosidase, while thewild type is inverting. Glycosidases having a double displacement mechanism oftencatalyze the transglycosylation to synthesize larger saccharide molecules. In order todetect the transglycosylation reaction in the T26H mutant T4L, several reaction con-

KUROKI et al.: T4 PHAGE LYSOZYME 363

FIGURE 1. Schematic view of the substrate and the products. The substrate used is the dimerof N-acetyl glucosaminyl N-acetyl muramic acid, in which the lactyl group is modified by D-alanyl-D-glutaminyl-diaminopimelic acid and the reducing end is reduced.

ditions were used. The ratio of the substrate to the mutant T4L was varied from 1:100to 1:1000, and the reaction temperatures used were 0 and 25 °C. At 25 °C, the sub-strate was hydrolyzed only to two muramyl peptides (NAG-NAM-DAla-DGlu-di-aminopimelic acid). One has a reduced reducing end (peak 1) and the other has a newreducing end with a �-anomeric configuration (peak 2) as described previously.9

However, when the reaction was performed at 0 °C, the T26H mutant hydrolyzed thesubstrate to at least five products (FIGURE 2), including the two muramyl peptidepeaks observed previously.9 In FIGURE 2, a representative chromatogram after a 60-min digestion of 0.1 mM substrate with 20 �M T26H mutant at pH 5.0 and 0 °C isshown. All peaks (peaks 1–6), including two new peaks, 5 and 6, were collected andidentified by electron ion spray mass spectrometry (ES-MS) as listed in TABLE 1. Itwas found that peaks 5 and 6 have masses of 2571.2 Da and 3422.4 Da, correspond-ing to the trimer [(GMP3)2-GMP3r] and tetramer [(GMP3)3-GMP3r] of the muramylpeptide having a reduced reducing end (FIGURE 1 and TABLE 1). The substrate waspartially hydrolyzed to a monomer, but also produced trimers, tetramers, and proba-bly pentamers of NAG-NAM-DAla-DGln-diaminopimelic acid. It is clear that themutant T26H catalyzes the transglycosylation reaction effectively.

The transglycosylation seen in the mutant T26H was quite effective with a ratio ofhydrolysis to transglycosylation of 1:10 after a 60-min reaction. This indicates thatthe covalent intermediate is more accessible to the saccharide than to the bulk water.Compounds such as the saccharyl or phenyl derivative, known as an acceptor groupfor transglycosylation, are relatively larger and more hydrophobic than a water mole-cule. The hydrophobicity of the acceptor binding site may decrease the accessibilityof the intermediate to water. The formation of the covalent intermediate also allows

ANNALS NEW YORK ACADEMY OF SCIENCES364

FIGURE 2. Elution pattern of the reaction product after digestion of substrate with the mutantT4L (T26H). The numbers shown in this figure represent the mass of each peak.

sufficient time for another saccharide to access the covalent intermediate, leading totransglycosylation.

REFERENCES

1. BLAKE, C. C. F., L. N. JOHNSON, G. A. MAIR, A. C. T. NORTH, D. C. PHILLIPS & V. R. SAR-MA. 1967. Proc. R. Soc. Lond. B167: 378–388.

2. GRÜTTER, M. G., L. H. WEAVER & B. W. MATTHEWS. 1983. Nature (Lond.) 303: 828–831.3. MATTHEWS, B. W. & S. J. REMINGTON. 1974. Proc. Natl. Acad. Sci. U.S.A. 71: 4178–4182.4. SVENSSON, B. & M. SØGAARD. 1993. J. Biotechnol. 29: 1–37.5. MALCOM, B. A., S. ROSENBERG, M. J. COREY, J. S. ALLEN, A. DE BAETSELIER & J. K.

KIRSCH. 1989. Proc. Natl. Acad. Sci. U.S.A. 86: 133–137.6. HARDY, L. W. & A. R. POTEETE. 1991. Biochemistry 30: 9457–9463.7. WEAVER, L. H., M. G. GRÜTTER & B. W. MATTHEWS. 1995. J. Mol. Biol. 245: 54–68.8. KUROKI, R., L. H. WEAVER & B. W. MATTHEWS. 1993. Science 262: 2030–2033.9. POTEETE, A. R., S. DAO-PIN, H. NICHOLSON & B. W. MATTHEWS. 1991. Biochemistry 30:

1425–1432.10. MUCHMORE, D. C., L. P. MCINTOSH, C. B. RUSSELL, D. E. ANDERSON & F. W. DAHLQUIST.

1989. Methods Enzymol. 177: 44–73.

KUROKI et al.: T4 PHAGE LYSOZYME 365

TABLE 1. Observed and Theoretical Mass of the Peaks Eluted from Reversed-PhaseHPLC

Peak Observed Theoretical Formulaa

1 870.5 870 GMP3r2 868.5 868 �-GMP33 868.5 868 �-GMP34 1721.6 1720 (GMP3)1–GMP3r (substrate)5 2571.2 2570 (GMP3)2–GMP3r6 3422.4 3420 (GMP3)3–GMP3r

aAbbreviations—GMP3: N-acetyl glucosaminyl N-acetyl muramic acid with D-Ala-D-Glu-diaminopimelic acid; GMP3r: the reducing end of GMP3 is reduced.