Role of Arginine Residues in the Active Site of the Membrane-Bound Lytic Transglycosylase B from Pseudomonas aeruginosa
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- 作者：Christopher W. Reid ; Neil T. Blackburn ; and Anthony J. Clarke
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：February 21, 2006
- 年：2006
- 卷：45
- 期：7
- 页码：2129 - 2138
- 全文大小：704K
- 年卷期：v.45,no.7(February 21, 2006)
- ISSN：1520-4995
文摘
Lytic transglycosylases cleave the 
-(1
4)-glycosidic bond in the bacterial cell wallheteropolymer peptidoglycan between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine(GlcNAc) residues with the concomitant formation of a 1,6-anhydromuramoyl residue. On the basis ofboth sequence alignments with and structural considerations of soluble lytic transglycosylase Slt35 fromEscherichia coli, four residues were predicted to be involved in substrate binding at the -1 subsite in thesoluble derivative of Pseudomonas aeruginosa membrane-bound lytic transglycosylase MltB. These residueswere targeted for site-specific replacement, and the effect on substrate binding and catalysis was determined.The residues Arg187 and Arg188, believed to be involved in binding the stem peptide on MurNAc, wereshown to play an important role in substrate binding, as evidenced by peptidoglycan affinity assays andSUPREX analysis using MurNAc-dipeptide as ligand. The Michaelis-Menten parameters were determinedfor the respective mutants using insoluble peptidoglycan as substrate. In addition to affecting the steady-state binding of ligand to enzyme, as indicated by increases in KM values, significant decreases in kcatvalues suggested that replacement of either Arg187 and Arg188 with alanine perturbed the stabilizationof both the transition state(s) and reaction intermediate. Thus, it appears that Arg187 and Arg188 arevital for proper orientation of the substrate in the active site, and furthermore this supports the proposedrole of the stem peptide at binding subsite -2 in catalysis. Replacement of Gln100, a residue that wouldappear to interact with the N-acetyl group on MurNAc, did not show any changes in substrate affinity oractivity.
-(14)-glycosidic bond in the bacterial cell wallheteropolymer peptidoglycan between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine(GlcNAc) residues with the concomitant formation of a 1,6-anhydromuramoyl residue. On the basis ofboth sequence alignments with and structural considerations of soluble lytic transglycosylase Slt35 fromEscherichia coli, four residues were predicted to be involved in substrate binding at the -1 subsite in thesoluble derivative of Pseudomonas aeruginosa membrane-bound lytic transglycosylase MltB. These residueswere targeted for site-specific replacement, and the effect on substrate binding and catalysis was determined.The residues Arg187 and Arg188, believed to be involved in binding the stem peptide on MurNAc, wereshown to play an important role in substrate binding, as evidenced by peptidoglycan affinity assays andSUPREX analysis using MurNAc-dipeptide as ligand. The Michaelis-Menten parameters were determinedfor the respective mutants using insoluble peptidoglycan as substrate. In addition to affecting the steady-state binding of ligand to enzyme, as indicated by increases in KM values, significant decreases in kcatvalues suggested that replacement of either Arg187 and Arg188 with alanine perturbed the stabilizationof both the transition state(s) and reaction intermediate. Thus, it appears that Arg187 and Arg188 arevital for proper orientation of the substrate in the active site, and furthermore this supports the proposedrole of the stem peptide at binding subsite -2 in catalysis. Replacement of Gln100, a residue that wouldappear to interact with the N-acetyl group on MurNAc, did not show any changes in substrate affinity oractivity.