Reactions of Second Stage of Biosynthesis of Teichuronic Acid of Micrococcus lysodeikticus Cell Walls *

A polysaccharide of alternating glucose and N-acetylmannosaminuronic acid residues is formed in the second stage of teichuronic acid biosynthesis by the particulate enzyme fraction of Micrococcus lysodeikticus. Di(N-acetylmannosaminuronylj-N-acetylglucosaminyl carrier lipid, the third intermediate formed in the initial stage of teichuronic acid synthesis, serves as an acceptor for the sequential and alternating addition of glucose residues from uridine diphosphate glucose and N-acetylmannosaminuronic acid residues from uridine diphosphate N-acetylmannosaminuronic acid. Glucosyl di (N acetylmannosaminuronyl j-N acetylglucosaminyl carrier lipid and N-acetylmannosaminuronylglucosyl-di(N-acetylmannosaminuronyl) N-acetylglucosaminyl carrier lipid are intermediates formed by the action of the glucosyltransferase and the N-acetylmannosaminuronyltransferase, respectively. Both uridine diphosphate glucose and uridine diphosphate N-acetylmannosaminuronic acid produce uridine diphosphate in an amount stoichiometric with the incorporation of monosaccharide residues into teichuronic acid. The transferases which catalyze the second stage of teichuronic acid synthesis are solubilized from the particulate enzyme fraction by Triton X-100. The glucosyltransferase can be selectively released at 5 to 10 mM magnesium ion, whereas at a concentration of 1 mM or less the N-acetylmannosaminuronyltransferase is also solubilized.

A polysaccharide of alternating glucose and N-acetylmannosaminuronic acid residues is formed in the second stage of teichuronic acid biosynthesis by the particulate enzyme fraction of Micrococcus lysodeikticus.
Di(N-acetylmannosaminuronylj-N-acetylglucosaminyl carrier lipid, the third intermediate formed in the initial stage of teichuronic acid synthesis, serves as an acceptor for the sequential and alternating addition of glucose residues from uridine diphosphate glucose and N-acetylmannosaminuronic acid residues from uridine diphosphate N-acetylmannosaminuronic acid. Glucosyl -di (N -acetylmannosaminuronyl j-N -acetylglucosaminyl carrier lipid and N-acetylmannosaminuronylglucosyl-di(N-acetylmannosaminuronyl) -N-acetylglucosaminyl carrier lipid are intermediates formed by the action of the glucosyltransferase and the N-acetylmannosaminuronyltransferase, respectively. Both uridine diphosphate glucose and uridine diphosphate N-acetylmannosaminuronic acid produce uridine diphosphate in an amount stoichiometric with the incorporation of monosaccharide residues into teichuronic acid. The transferases which catalyze the second stage of teichuronic acid synthesis are solubilized from the particulate enzyme fraction by Triton X-100. The glucosyltransferase can be selectively released at 5 to 10 mM magnesium ion, whereas at a concentration of 1 mM or less the N-acetylmannosaminuronyltransferase is also solubilized.

Sequential
Addition of Glucose and N-Acetylmannosaminuronic Acid to Component C -Since Component C is formed in reaction mixtures which contain UDP-ManNAcUA but lack UDP-glucose, the first step in the second stage of teichuronic acid synthesis should utilize UDP-glucose. To demonstrate sequential addition of first a glucose residue and then an N- acetylmannosaminuronic acid residue to Component C, the following three reaction mixtures were prepared. In reaction mixture A, the particulate enzyme fraction was incubated with UDP-GlcNAc and [G-WIUDP-ManNAcUA to make [YJManNAcUA-labeled Component C. In reaction mixture B, unlabeled Component C was prepared and subsequently incubated with only UDP-[Wlglucose.
In reaction mixture C, the two initial incubations were the same as in reaction mixture B but with unlabeled substrates and the final incubation was with only [G-W]UDP-ManNAcUA.
Following each incubation whether with unlabeled or with labeled substrates, the product. particulate enzyme complex was recovered by sedimentation, resuspended, and again recovered by sedimentation to remove residual substrates. Each labeled complex was subjected to paper chromatography in solvent System A. Fig. 4 shows radiochromatogram tracings obtained from reaction mixtures A, B, and C in Frames A, B, and C, respectively. Comparison of the chromatographic mobility of the major radioactive products with an internal marker of uracil indicates that the sequential addition of the labeled residues of glucose and N-acetylmannosaminuronic acid yielded products with progressively decreased mobility relative to Component C. The product of Reaction B is presumed to be Glc-(Man-NAcUA),-GlcNAc-phosphoundecaprenol (Component D) and that of Reaction C is presumed to be ManNAcUA-Glc-(Man-NAcUA),-GlcNAc-phosphoundecaprenol (Component E). The addition of N-acetylmannosaminuronic acid to Component C is dependent upon the prior addition of glucose, since little product of the mobility of Component E was observed in Reaction A which had been incubated with [G-WIUDP-Man-NAcUA.  to N-acetylmannosaminuronic acid and glucose residues indicates that the value for n is about 40 (cf. Figs. 1 and 2). This value is the same as that reported for teichuronic acid isolated from cell walls (10).
Results reported here show that Component C labeled with either N-acetylglucosamine or N-acetylmannosaminuronic acid is incorporated into teichuronic acid under conditions appropriate for the second stage of teichuronic acid synthesis. Neither paper chromatography nor gel filtration in the presence of detergent separated the label introduced as Component C from the teichuronic acid synthesized in its presence, although unreacted Component C was readily distinguished from teichuronic acid by both procedures. This evidence indicates that Component C serves as an acceptor for the assembly of the glucose-containing portion of teichuronic acid. The sequential addition of first glucose and then N-acetylmannosaminuronic acid to Component C, yielding materials designated as Glc-(ManNAcUA),-GlcNAc carrier lipid (Component D) and ManNAcUA-Glc-(ManNAcUA),-GlcNAc carrier lipid (Component E), provides additional evidence that Component C functions as an acceptor in the second stage of teichuronic acid synthesis.
The results presented here provide no evidence to indicate that the transfer of glucose from UDP-glucose to Component C or in the subsequent elongation of the product polysaccharide with N-acetylmannosaminuronic acid and glucose involves any carrier lipid other than that already present in Component C. The sequential addition of glucose and N-acetylmannosaminuronic acid residues suggests that each saccharide is incorporated 1 residue at a time alternately by two transferases. Each transferase has a specificity for the donor nucleotide and for the acceptor which must have the alternate saccharide as the nonreducing terminal residue. The solubiliza-tion of the glucosyltransferase and the N-acetylmannosaminuronyltransferase supports the concept of alternate addition of single residues. Complete verification must await purification of both transferases and demonstration that the two purified enzymes act sequentially. The experimental results eliminate an alternate mechanism by which a mixed disaccharide might form, possibly as a derivative of a carrier lipid, and subsequently polymerize to yield a heteropolysaccharide having a sequence of alternating saccharide residues. The data presented here do not indicate whether the carrier lipid moiety of Component C (undecaprenol monophosphate) is incorporated into the final in. vitro reaction product. If still attached to the carrier lipid, the in vitro product might be the precursor for the attachment of fully assembled polysaccharide onto the peptidoglycan to form cell wall which is the ultimate product of the biosynthetic pathway. Such a transfer reaction might not occur in the in vitro system catalyzed by the particulate enzyme fraction for lack of an appropriate final acceptor.
If the teichuronic acid formed in vitro is ultimately transferred from carrier lipid to peptidoglycan, the trisaccharide portion formed by the reactions of the initial stage of teichuronic acid synthesis must become part of a linkage region by which the polysaccharide of glucose and N-acetylmannosaminuronic acid is covalently attached to peptidoglycan.
The structure of the linkage between teichuronic acid and peptidoglycan in native cell walls is not yet fully established. Nasirud-din and Jeanloz (10) have proposed attachment of teichuronic acid from a glucose residue through phosphate to C-6 of an N-acetylmuramic acid residue of peptidoglycan. In contrast, our results suggest that the teichuronic acid is attached through the saccharide residues of Component C. TWO groups have reported an excess of hexosamine residues in the teichuronic acid isolated from cell walls following enzymatic digestion of the peptidoglycan (11, 12) which is consistent with the linkage region suggested by our biosynthetic studies.