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• Author: Anthony J. Clarke
• Responsible Curator: Anthony J. Clarke

Substrate specificities

Figure 1. Reaction catalyzed by family GH103 enzymes. LT, lytic transglycosylase. (click to enlarge).

Kinetics and Mechanism

The lytic transglycosidases, strictly speaking, are retaining enzymes. However, they are not hydrolases but rather catalyse an intramolecular glycosyl transfer reaction onto the C-6 hydroxyl group of the muramoyl residue leading to the generation of a terminal 1,6-anhydromuramoyl product (Figure 1) that lacks a reducing end [3]. No detailed analyses involving both steady state and pre-steady state kinetic studies have been reported, but the Michaelis-Menten (K_M and V_max) parameters have been estimated for Pseudomonas aeruginosa MltB acting on insoluble peptidoglycan sacculi [4].

Catalytic Residues

Figure 2. Reaction mechanism proposed for E. coli and P. aeruginosa MltB. (click to enlarge).

Examination of crystal structures of E. coli Slt35 (a soluble proteolytic derivative of MltB) and theoretical considerations led to the proposal of a mechanism that accommodates a single catalytic residue at its active site. Thus, based on the complexes formed with murodipeptide, chitobiose, and the inhibitor bulgecin, a two-step neighboring group participation mechanism involving substrate-assisted catalysis has been invoked analogous to the family GH18 chitinases and chitobiases, family GH20 N-acetyl-β-hexosaminidases, and family GH23 lytic transglycosylases [6]. Thus, the catalytic residue Glu162 is proposed to serve initially as a general acid to donate a proton to the glycosidic oxygen of the linkage to be cleaved. At the same time the MurNAc 2-acetamido group acts as a nucleophile and attacks the anomeric centre. The transition state leading to the intermediate possesses oxocarbenium ion character (Figure 2). In the second step abstraction of the C-6 hydroxyl proton of the oxazolinium species by Glu162 which now serves as a general base leads to nucleophilic attack and the formation of 1,6-anhydromuramic acid product, again through a transition state with oxocarbenium ion character. The β-hexosaminidase inhibitor NAG-thiazoline (Figure 2) was found to inhibit P. aeruginosa MltB thus supporting the proposal for the formation of an oxazolinium ion intermediate [7], and the results of a site-directed mutagenesis study suggest that Ser216 orients the N-acetyl group on MurNAc at the -1 subsite of MltB for its participation in a substrate-assisted mechanism of action [8].

Three-dimensional structures

Three-dimensional structures are available for several family GH103 enzymes, the first solved being that of E. coli MltB (Slt35) [5]. The catalytic domain of the enyzmes possesses the well characterized α+β "lysozyme fold."

Family Firsts

First identification of lytic transglycosylase

MltB from E. coli [2].

First catalytic nucleophile identification

Uses neighboring group participation mechanism.

First general acid/base residue identification

Inferred by X-ray crystallography of E. coli MltB [5].

First 3-D structure

E. coli MltB [5].

First identification as a lipoprotein

E. coli MltB [9].

First identification of localization to outer membrane

E. coli MltB [9].

Frist demonstration of molecular interactions between GH103 enzymes and penicillin-binding proteins

E. coli MltB [10].

References

1. Blackburn NT and Clarke AJ. Identification of four families of peptidoglycan lytic transglycosylases. J Mol Evol 2001 Jan; 52(1) 78-84. pmid:11139297. PubMed HubMed [1]
2. Engel H, Smink AJ, van Wijngaarden L, and Keck W. Murein-metabolizing enzymes from Escherichia coli: existence of a second lytic transglycosylase. J Bacteriol 1992 Oct; 174(20) 6394-403. pmid:1356966. PubMed HubMed [2]
3. Höltje JV, Mirelman D, Sharon N, and Schwarz U. Novel type of murein transglycosylase in Escherichia coli. J Bacteriol 1975 Dec; 124(3) 1067-76. pmid:357. PubMed HubMed [3]
4. Blackburn NT and Clarke AJ. Characterization of soluble and membrane-bound family 3 lytic transglycosylases from Pseudomonas aeruginosa. Biochemistry 2002 Jan 22; 41(3) 1001-13. pmid:11790124. PubMed HubMed [4]
5. van Asselt EJ, Dijkstra AJ, Kalk KH, Takacs B, Keck W, and Dijkstra BW. Crystal structure of Escherichia coli lytic transglycosylase Slt35 reveals a lysozyme-like catalytic domain with an EF-hand. Structure 1999 Oct 15; 7(10) 1167-80. pmid:10545329. PubMed HubMed [5]
6. van Asselt EJ, Kalk KH, and Dijkstra BW. Crystallographic studies of the interactions of Escherichia coli lytic transglycosylase Slt35 with peptidoglycan. Biochemistry 2000 Feb 29; 39(8) 1924-34. pmid:10684641. PubMed HubMed [6]
7. Reid CW, Blackburn NT, Legaree BA, Auzanneau FI, and Clarke AJ. Inhibition of membrane-bound lytic transglycosylase B by NAG-thiazoline. FEBS Lett 2004 Sep 10; 574(1-3) 73-9. doi:10.1016/j.febslet.2004.08.006 pmid:15358542. PubMed HubMed [7]
8. Reid CW, Legaree BA, and Clarke AJ. Role of Ser216 in the mechanism of action of membrane-bound lytic transglycosylase B: further evidence for substrate-assisted catalysis. FEBS Lett 2007 Oct 16; 581(25) 4988-92. doi:10.1016/j.febslet.2007.09.037 pmid:17910958. PubMed HubMed [8]
9. Ehlert K, Höltje JV, and Templin MF. Cloning and expression of a murein hydrolase lipoprotein from Escherichia coli. Mol Microbiol 1995 May; 16(4) 761-8. pmid:7476170. PubMed HubMed [9]
10. von Rechenberg M, Ursinus A, and Höltje JV. Affinity chromatography as a means to study multienzyme complexes involved in murein synthesis. Microb Drug Resist 1996 Spring; 2(1) 155-7. pmid:9158739. PubMed HubMed [10]