Characterization of Wall Teichoic Acid Degradation by the Bacteriophage ϕ29 Appendage Protein GP12 Using Synthetic Substrate Analogs*

Background: The GP12 protein from bacteriophage ϕ29 is a likely wall teichoic acid hydrolase. Results: GP12 is an efficient catalyst of wall teichoic acid hydrolysis that is influenced by glycosylation of the wall teichioc acid polymer. Conclusion: GP12 may serve as a tool for the characterization of wall teichoic acid polymers. Significance: Characterization of a prototypical wall teichoic acid hydrolase using chemically defined substrates. The genetics and enzymology of the biosynthesis of wall teichoic acid have been the extensively studied, however, comparatively little is known regarding the enzymatic degradation of this biological polymer. The GP12 protein from the Bacillus subtilis bacteriophage ϕ29 has been implicated as a wall teichoic acid hydrolase. We have studied the wall teichoic acid hydrolase activity of pure, recombinant GP12 using chemically defined wall teichoic acid analogs. The GP12 protein had potent wall teichoic acid hydrolytic activity in vitro and demonstrated ∼13-fold kinetic preference for glycosylated poly(glycerol phosphate) teichoic acid compared with non-glycosylated. Product distribution patterns suggested that the degradation of glycosylated polymers proceeded from the hydroxyl terminus of the polymer, whereas hydrolysis occurred at random sites in the non-glycosylated polymer. In addition, we present evidence that the GP12 protein possesses both phosphodiesterase and phosphomonoesterase activities.

The genetics and enzymology of the biosynthesis of wall teichoic acid have been the extensively studied, however, comparatively little is known regarding the enzymatic degradation of this biological polymer. The GP12 protein from the Bacillus subtilis bacteriophage 29 has been implicated as a wall teichoic acid hydrolase. We have studied the wall teichoic acid hydrolase activity of pure, recombinant GP12 using chemically defined wall teichoic acid analogs. The GP12 protein had potent wall teichoic acid hydrolytic activity in vitro and demonstrated ϳ13fold kinetic preference for glycosylated poly(glycerol phosphate) teichoic acid compared with non-glycosylated. Product distribution patterns suggested that the degradation of glycosylated polymers proceeded from the hydroxyl terminus of the polymer, whereas hydrolysis occurred at random sites in the non-glycosylated polymer. In addition, we present evidence that the GP12 protein possesses both phosphodiesterase and phosphomonoesterase activities.
Wall teichoic acids (WTAs) 2 are polyanionic glycopolymers covalently attached to peptidoglycan in the cell wall of Grampositive bacteria. Prototypical WTA polymers are repeating units of 1-3-linked glycerol phosphate or 1-5-linked ribitol phosphate and constitute roughly 50% of the wall mass (1). WTAs have been associated with diverse cellular processes including cell division and morphology, peptidoglycan assembly, bacteriophage recognition and adherence, and as a virulence determinant (1). Recently, the disruption of WTA biosynthesis was shown to restore the susceptibility of methicillin-resistant Staphylococcus aureus to certain ␤-lactam anti-biotics (2)(3)(4), revealing a surprising role for WTA in ␤-lactam resistance and highlighting steps in its biosynthetic pathway as attractive antibacterial therapeutic targets.
The genetics and biochemistry of WTA biosynthesis have been extensively studied in both the model Gram-positive bacterium Bacillus subtilis and the pathogen S. aureus (1). Biosynthesis of poly(glycerol phosphate) WTA in B. subtilis is illustrative (see Scheme 1). Polymer synthesis begins on the inner cell surface with the addition of N-acetlyglucosamine-1-phosphate to membrane-bound undecaprenyl phosphate, catalyzed by the TagO enzyme and generating lipid ␣ (WTA intermediates are named after the enzymes that act on them (5), summarized in Table 1). Next, TagA adds N-acetlymannosamine to lipid ␣ to form lipid ␤, the substrate for the primase enzyme TagB that adds a single glycerol phosphate molecule to form lipid .1 (6). TagF is the polymerase that adds 30 to 50 glycerol phosphate units to this substrate (7), followed by the modification of multiple glycerol units in poly(glycerol phosphate) WTA with ␣-glucose by TagE (8). The polymer is then transported to the extracellular surface by the TagGH ABC transporter system (9) where the gene products of the dltABCD operon effect D-alalynation (10) and the tagTUV enzymes are believed to catalyze final polymer transfer to N-acetylmuramic acid of peptidoglycan (11).
In contrast to the thorough understanding of its biosynthesis, the enzymology and biological relevance of WTA degradation have not been well studied. Evidence of such enzymatic activity was first reported more than 40 years ago by Wise and co-workers (12), with the discovery that B. subtilis is capable of utilizing its cell wall as a phosphate source. This was attributed to expression of a so-called teichoicase with glycerophosphodiesterase activity specific for WTA. It was later speculated that the expression of this enzyme may be induced by phosphate starvation (13), enabling the procurement of necessary phosphate from WTA. In line with this, during phosphate-limited growth of B. subtilis, much of the WTA in the cell wall is replaced by the phosphate-free polymer teichuronic acid (14,15). Apart from the potential importance in the phosphate starvation response, a role for WTA degradation in the regulation of cell wall structure and composition can be envisioned. For instance, WTAs have been shown to direct bacterial peptidoglycan hydrolases (16 -19) and consequently, influence wall remodeling. Also, as much as 50% of the wall is recycled during bacterial cell division (20,21), implying a need perhaps for enzymes capable of processing the very large quantities of WTA in peptidoglycan.
Through activity-guided purification, Kusser and Fielder (22,23) achieved some enrichment of the wall teichoicase activity from B. subtilis and later from Bacillus pumilus (24), yet these studies were limited in two important aspects. First, the enzyme preparations were never linked to a gene or protein sequence. Second, the substrates used were poorly defined, consisting of crude cell wall preparations or partially purified WTAs. Thus, bacterial teichoicases remain obscure, as are the mechanistic details of their biochemical activity.
Although bacterial enzymes that degrade WTA and their corresponding genes have yet to be identified in any bacterium, the GP12 protein produced by the B. subtilis bacteriophage 29 is thought to possess this activity. GP12 is an appendage protein found in the neck region of 29 that is involved in host cell recognition and entry (25). During phage maturation, GP12 forms homotrimers that undergo autoproteolysis mediated by an intramolecular chaperone domain (26), prior to forming the 12 appendages attached to the neck of phage particles (25,27). As part of a crystallographic investigation of this large (92 kDa) multifunctional protein, Xiang et al. (27) showed that GP12 could drastically reduce the apparent molecular size of poly-(glycerol phosphate) WTA extracted from B. subtilis 168, sug-gesting that the protein has inherent wall teichoicase activity. The activity was noted to be highly dependent on divalent metal ions, two of which (Ca 2ϩ and Mg 2ϩ ) were observed bound at the proposed WTA-degrading active site in a pectin lyase domain (28), prompting the suggestion of a two-metal ion mechanism for catalysis of WTA phosphodiester bond hydrolysis (27). Analogous mechanisms for phosphate ester hydrolysis involving a bimetallated active center are known for enzymes from the binuclear metallophosphatase family, which includes glycerophosphodiesterases and purple acid phosphatases (29), and interestingly, GP12 is structurally unrelated to this family.
In light of the previous qualitative examination, GP12 represents an ideal candidate for the study of enzyme-catalyzed WTA degradation. To that end, we have performed in-depth investigations on this prototypic wall teichoicase (GP12) using chemically defined and soluble WTA substrate analogs. Indeed, the study of WTA biosynthetic enzymes has been greatly enabled by chemoenzymatic syntheses of analogs for the biosynthetic intermediates. In place of the 55-carbon undecaprenyl chain, such analogs feature a more tractable lipid moiety that facilitates manipulation in aqueous systems (8, 30 -33). Moreover, soluble poly(glycerol phosphate) polymer is accessible through the polymerization of glycerol phosphate onto CDP-glycerol (34). Equipped with chemically defined substrates, we developed HPLC-based assays for poly(glycerol phosphate) hydrolysis that were used to meticulously characterize the wall teichoicase activity of GP12. We found that polymer glycosylation led to an enhanced rate of polymer degrada-  tion, and also appeared to influence the mode of GP12 action. Further interrogation of GP12 catalytic activity suggested that the enzyme utilizes phosphodiesterase and phosphomonoesterase activity in its reaction with WTA polymers.
Cloning, Expression, and Purification of GP12-The genetic sequence for a GP12 construct missing the first 88 amino acids was PCR-amplified from pET28b-GP12 (27) using primers ⌬N88GP12-Forward and GP12-Reverse (supplemental Table  S1). The amplicon was cloned into pET28b plasmid using NcoI and HindIII restriction sites to generate p⌬N88GP12. Escherichia coli BL21-CodonPlus (DE3)-RIL transformed with p⌬N88GP12 was grown at 25°C in Luria Bertani (LB) media supplemented with 20 g/ml of chloramphenicol and 50 g/ml of kanamycin to an A 600 of 0.6 before inducing protein expression with 0.1 mM isopropyl ␤-D-1-thiogalactopyranoside. Expression was continued for 20 h at 16°C. Cells were harvested by centrifugation (8500 ϫ g for 15 min at 4°C) and washed with 0.85% NaCl, prior to resuspension in a buffer containing Tris-HCl (pH 7.5), 300 mM NaCl, 25 mM imidazole. EDTA-free Protease Inhibitor Mixture (Roche Diagnostics, Laval, Québec, Canada) was added to the suspension and the cells were lysed by two consecutive passages through a cell disruptor (Constant Systems, Daventry, Northants, UK) at 30,000 p.s.i. Cell lysates were clarified by centrifugation (40,000 ϫ g for 60 min at 4°C) prior to loading on a 1-ml HisTrapHP column (GE Life Sciences) pre-equilibrated with resuspension buffer. GP12 was eluted over a 25 to 400 mM imidazole gradient, and fractions containing pure protein were pooled and the buffer exchanged for 50 mM Tris (pH 7.5), 100 mM NaCl, 15% glycerol (v/v), using a HiPrep 26/10 desalting column (GE Life Sciences). Protein concentration was determined according to the method of Gill and von Hippel (36).
Synthesis of WTA Analog Precursors-Lipid ␣, ␤, and .1 analogs were synthesized as described previously (30,32). and at 271 nm for cytidine containing reagents) by reversed phase HPLC using a Waters HPLC system (Waters 1525 HPLC pump and 2998 PDA detector; Waters, Mississauga, Ontario, Canada), or by scintillation counting with an in-line 150TR Flow Scintillation Analyzer (PerkinElmer). Upon reaction completion, enzymes were removed using a centrifugal filter with a 10,000 Da molecular mass cut-off (Pall Life Sciences, Mississauga, Ontario, Canada).
Synthesis of WTA Analogs-The chemoenzymatic approach for the preparation of soluble tricecyl-linked WTA analogs is summarized in Scheme 1. Lipid .40 analog was synthesized using TagF according to established methods, with the lipid .1 analog and CDP-glycerol as the glycerol phosphate acceptor and donor, respectively (32,33). To incorporate 14 C into the glycerol phosphate polymer chain, 0.91 Ci of CDP-[U-14 C]glycerol (98.7 mCi/mmol) was included in the reaction and the analogswere prepared at 91 mCi/mmol. [ 14 C]ManNAc lipid .40 analog was similarly prepared at 20 mCi/mmol, by the inclusion of 0.2 Ci of [ 14 C]ManNAc lipid .1 analog to the reaction. Reaction progress was monitored by anion exchange HPLC using a DNA PAC PA200 anion exchange column (Thermo Fisher Scientific), with visualization by in-line scintillation counting. The solvents used were: A, 20 mM ammonium bicarbonate (pH 8.0) containing 10% acetonitrile (v/v) and B, 20 mM ammonium bicarbonate (pH 8.0) containing 1 M NaCl and 10% acetonitrile (v/v). Substrates and products were separated over the following gradient condition for solvent B: 0 -30% (3 min), 30 -45% (10 min), 45-100% (5 min), and finally 100% for 5 min. Upon reaction completion, enzymes and low molecular weight by-products were removed using centrifugal filters with molecular mass cut-offs of 30,000 and 3000 Da, respectively (Pall Life Sciences).
Polymer glycosylation reactions were performed as described previously (8), by the incubation of TagE with [ 14 C]glycerol-or [ 14 C]ManNAc lipid .40 analog and a molar excess of UDP-glucose to glycerol phosphate units, or likewise with nonradioactive lipid .40 analog and UDP-[ 14 C]glucose. The progress of glycosylation reactions was assessed by anion exchange HPLC using chromatographic conditions identical to those for the non-glycosylated analog, or by UDP-glucose consumption with ion-paired reversed phase HPLC (8). Upon reaction completion, enzymes and reaction by-products were removed by ultrafiltration as above.
Wall Teichoic Acid Degradation Assays-Unless otherwise stated, GP12 reactions with WTA analogs were performed at room temperature in a buffer comprised of 50 mM Tris-HCl (pH 7.5) and 30 mM MgCl 2 . Reactions were initiated by addition of GP12 and terminated at specified time points by the addition of urea to a final concentration of 6 M. Radioactive reaction products were separated by anion exchange HPLC as described above. Initial rate analyses for GP12 degradation of 14 C-labeled WTA analogs were performed with varying concentrations of GP12 and fixed amounts of WTA substrate. The amount of fully polymeric starting substrate remaining after specific time points was quantified by peak integration.
Liquid Chromatography-Mass Spectrometry-Reactions (30 l) between GP12 and non-radioactive lipid .40 analogs were quenched with an equal volume of methanol, and product mix-tures were analyzed by normal phase liquid chromatography ESI-QTOF mass spectrometry, as described previously (37).
GP12 Hydrolysis of para-Nitrophenyl Phosphates-The catalytic activity of GP12 with pNPP or bis-pNPP was assayed by monitoring the production of para-nitrophenol (pNP). Unless otherwise stated, reactions (100 l) were performed in buffer containing 50 mM Tris-HCl (pH 7.5), 30 mM MgCl 2 , and 10 mM CaCl 2 . After initiation with GP12, the absorbance at 400 nm was measured continuously on a SpectraMax microplate reader (Molecular Devices, Sunnyvale, CA). pNP generated was quantified by interpolation to a standard curve.
Assays for the determination of kinetic parameters contained substrate concentrations ranging between 40.0 and 0.625 mM for pNPP, and from 28.50 to 0.44 mM for bis-pNPP. Initial rates of pNP production (substrate consumption Ͻ10%) were plotted as a function of substrate concentration and the data fit by non-linear regression (Prism 6.0; GraphPad, La Jolla, Measurement of Inorganic Phosphate-Reactions (50 l) of GP12 with WTA analogs or bis-pNPP were performed in a buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 and for bis-pNPP hydrolysis, 5 mM CaCl 2 . At specified time points, reactions were stopped by the addition of EDTA to a final concentration of 10 mM, and then 100 l of BIOMOL Green reagent (Enzo Life Sciences, Inc., Farmingdale, NY) was added. P i was detected by measuring absorbance at 640 nm after 15 min, and quantified by interpolation to a standard curve.

Results
Characterization of WTA Analogs-Aiming for a polymer of roughly native length at 40 glycerol phosphate units, we synthesized tridecyl-linked WTA analogs using TagF, and CDP-[ 14 C]glycerol as the glycerol phosphate donor at 39 times the molar concentration of lipid .1 acceptor. On completion of the reaction, anion exchange HPLC analysis showed a single radioactive product peak, indicating a polyanionic species formed from the polymerization of glycerol phosphate onto the lipid .1 analog (Fig. 1A). MALDI-TOF analyses of parallel non-radioactive reactions were consistent with the production of lipid .40 analog, but also showed the polymerization product to comprise a mixture of species differing successively by the mass of the repeating WTA polymer unit (supplemental Fig. S1). This mass distribution coincides with the previous description of non-processive polymerization catalyzed by TagF that precludes the generation of a completely homogenous polymer (33). By controlling the ratio of TagF substrates (e.g. lipid .1 analog:CDP-glycerol ratio of 1:39), we were able to target the product mixture to a relatively uniform range of species that co-eluted during anion exchange chromatography. The small differences in polymer sizes (1-5 glycerol phosphate units) contrasts favorably with far greater differences for purified WTA or WTA in cell wall extracts (typical polymer sizes of 30 -50 glycerol phosphate units) and would not adversely affect subsequent characterizations of the products from degradation reactions.
Glycosylation of [ 14 C]glycerol lipid .40 analog using TagE and UDP-D-glucose likewise yielded a single radioactive product, as judged by anion exchange HPLC (Fig. 1B). A product with an identical retention time was seen on reaction of nonradiolabeled lipid .40 analog with UDP-D-[ 14 C]glucose, confirming this species as glycosylated polymer (Fig. 1B). MALDI-TOF MS analysis indicated virtually complete modification of all available glycerol phosphate units with glucose (supplemental Fig. S1), also reflected by assessment of UDP-glucose consumed in the reaction (not shown).
TagF most efficiently elongates the primer lipid .1 (33), but is also capable of using CDP-glycerol as a primer for poly(glycerol phosphate) synthesis, forming CMP-poly(glycerol phosphate) (34). We prepared this nucleotide-linked polymer to test the generality of GP12 activity and verify that the tridecyl tail did not hamper enzymatic degradation. The reaction for [ 14 C]glycerol CMP-poly(glycerol phosphate) WTA showed a single product on anion exchange HPLC analysis (Fig. 1C) and as previously observed, this analog was estimated to exceed the polymer length of the lipid .40 analog (8,34). As with the tridecyl-linked analog, glycosylation of CMP-poly(glycerol phosphate) WTA yielded a single product with a shorter retention time than non-glycosylated (Fig. 1C).
GP12 Catalyzes the Degradation of WTA Analogs-With chemically defined [ 14 C]glycerol-labeled WTA substrates in hand, we examined the activity of GP12 with these analogs by monitoring product formation with anion exchange HPLC. Fig.  2, A and B, show the glycerol containing products generated from lipid .40 analogs over time, and it was evident that GP12 catalyzed cleavage of the glycerol phosphate polymer chain. Whereas products from the reaction with the non-glycosylated analog spanned a continuum of polymer sizes, a narrow range of products corresponding to species of reduced apparent polymer length was produced from reaction of the glycosylated analog. These emerged after as little as 2 min and were confirmed to contain glucose from a parallel reaction of [ 14 C]glucose-glycosylated lipid .40 analog (Fig. 2B). As well, degradation occurred to a greater extent and with lower GP12 concentrations for the glycosylated substrate. Under reaction conditions identical to those for WTA analogs, we detected no activity for GP12 with glycerol phosphate (phosphatase), CDP-glycerol (phosphodiesterase), or the lipid .1 analog (phosphodiesterase).
Similar results were obtained when CMP-poly(glycerol phosphate) WTAs were used as substrates, suggesting no significant influence of the tridecyl tail on activity (Figs. 3, A and  B). Subsequent investigations were focused on the tridecyllinked analogs, which could be quantified based on definitive knowledge of the starting concentration of glycerol phosphate acceptor and the polymer size.
GP12 More Efficiently Degrades Glycosylated Lipid .40 Analog-The above results implied GP12 was more active toward glycosylated WTA analogs. To explore this apparent preference, we examined activity as a function of time and GP12 concentration, monitoring substrate consumption with anion exchange HPLC. We note here that substrate concentrations were in large excess of GP12 and substrate consumption was limited to 15% to ensure measurement of initial rates and to favor turnover of the fully polymeric, time 0 substrate. The assay showed a linear response with respect to time and enzyme concentration and under these conditions, apparent turnover by GP12 was estimated at 6.2 and 80 min Ϫ1 for non-glycosylated and glycosylated lipid .40 analog, respectively (Fig. 4, A  and B).
Product Distribution Analyses-The degradation of non-glycosylated and glycosylated analogs seemed to produce different outcomes with respect to the size and distribution of products. We hypothesized that this was indicative of different modes of degradation for the two substrates. However, because the starting material in either case was labeled throughout the polymer chain, products without the tridecyl moiety were indistinguishable from those having this appendage. To differentiate between these and thereby gain insight into the mode of poly-mer degradation, we synthesized WTA analogs labeled with 14 C exclusively at ManNAc to ensure the detection of only tridecyl-containing products, as GP12 was not active on the lipid .1 moiety. Fig. 5A shows HPLC chromatograms of products from the reaction of GP12 with lipid .40 analog radioactively labeled as described above. Between 0 and 4 h, the product distribution resembled that from reactions with the [ 14 C]glycerol-labeled polymer. Further reaction yielded a product distribution skewed toward products with smaller apparent polymer sizes, as opposed to the relatively uniform distribution observed for [ 14 C]glycerol-labeled products. Hence, the broad variability in product composition from the lipid .40 analog reaction includes both tridecyl-linked and tridecyl-free species. In con-

Wall Teichoic Acid Degradation by GP12
JULY 31, 2015 • VOLUME 290 • NUMBER 31 trast, tridecyl-linked products, significantly reduced in apparent polymer size, were detected after 2 min of reaction with [ 14 C]ManNAc-glycosylated lipid .40 (Fig. 5B) and the product distribution remained skewed toward smaller species. Consideration of these findings alongside observations with the equivalent [ 14 C]glycerol-labeled analog suggests that these truncated, tridecyl-linked species were co-produced with 2-5-mers of (glucose) glycerol phosphate in the early stages of glycosylated lipid .40 analog degradation. Collectively, these results portray distinct modes of GP12 action against glycosylated and non-glycosylated substrates; the former is preferentially hydrolyzed at regular intervals, whereas hydrolysis occurs at random sites throughout the latter. Notably, degradation reactions ultimately generated tridecyl-linked products containing 2-5 monomer units, irrespective of polymer glycosylation. It follows that poly(glycerol phosphate) substrates of GP12 contain at least 3 monomer units.
LC-MS analyses of WTA degradation products offered additional support for the inferred modes of polymer degradation ( Table 2, supplemental Fig. S2). First, this analysis confirmed that cleavage of the polymer occurred at phosphate esters. Furthermore, various glycerol phosphate-containing species were identified from a reaction with the non-glycosylated analog, whereas species containing one or two (glycosylated) glycerol units were detected from a reaction with glycosylated WTA. Surprisingly, phosphomonoester products anticipated from phosphodiester hydrolysis were largely absent from the species detected, which was suggestive of monoesterase activity by GP12.
GP12 Phosphomonoesterase Activity and the Mechanism of Hydrolysis-The findings from LC-MS analyses were not fully representative of WTA degradation products apparent from the HPLC studies, likely because of low ion abundance and/or ion suppression in these experiments. We therefore sought to establish whether GP12 was additionally capable of catalyzing monoester hydrolysis by initially examining the products from extensive degradation of [ 14 C]glycerol WTA analogs. Dephosphorylated WTA monomers (glycerol, glucose-glycerol) were observed as major products from these reactions (Fig. 6A), and could have possibly arisen from hydrolysis of the corresponding phosphomonoesters generated in preceding phosphodiester hydrolysis. Alternatively, dephosohorylated WTA monomers  might be the product of diester hydrolysis, especially as glycerol phosphate was found not to be a substrate for GP12. To clarify, we queried whether P i was generated during GP12 reactions with WTA analogs under conditions commensurate with significant polymer degradation (see Figs. 2 and 5). The results of these experiments, presented in Fig. 6B, show that P i release was concomitant with polymer degradation, but was not stoi-chiometric relative to the input of phosphate in the form of phosphodiester linkages. This was consistent with the modes of action deduced for GP12, whereby not all available phosphodiester linkages are subject to catalytic hydrolysis. Moreover, higher P i output for the reaction of the glycosylated analog indicated the presence of increased amounts of hydrolyzable monoesters, as could be expected from the greater extent of

Ion observed (m/z) Species
Lipid .40 analog 171.02 ͓GroP͔ Ϫ 245.06 ͓Gro-P-Gro͔ Ϫ 399.07 ͓Gro-P-Gro-P-Gro͔ Ϫ 553.07 ͓Gro-P-Gro-P-Gro-P-Gro͔ Ϫ 707.07 ͓Gro-P-Gro-P-Gro-P-Gro-P-Gro͔ Ϫ 701. 16 ͓Gro-P-Gro-P-Gro-P-Gro-P-Gro-P-Gro-P-Gro-P-Gro-P-Gro͔ Ϫ2 ͓Gro(Glu)-P-Gro(Glu)͔ Ϫ degradation observed for this analog. We conclude from these findings that GP12 exercises phosphomonoesterase activity in situ, on products formed during ongoing WTA hydrolysis. The activity of GP12 against WTA substrates therefore appeared to involve two hydrolytic reactions, illustrated in Fig.  6C. The relative abundance of the products of phosphodiester cleavage (i.e. alcohol and phosphate monoester) was not clear from these reactions with polyphosphodiester WTA substrates, presenting a challenge to ascertaining the extent of phosphate ester hydrolysis. As a result, we employed bis-pNPP and pNPP as simple substrates to model the reactions catalyzed by GP12. On the basis of pNP liberation, GP12 was active on both molecules in an enzyme-concentration dependent man-ner that was stimulated by Ca 2ϩ (Fig. 7A). Enzyme turnover was greater with the diester-containing substrate and indeed, markedly higher k cat and k cat /K m were observed for bis-pNPP (Table  3). In general, the kinetic parameters reflected poor binding and modest turnover for either substrate, but were nonetheless illustrative of preferential diesterase activity by GP12. Hence, the hydrolysis of bis-pNPP by GP12 was predicted to yield equimolar amounts of pNPP and pNP in the initial stages of the reaction when [bis-pNPP] Ͼ Ͼ [pNPP]. To determine the end result of pNPP generated during bis-pNPP hydrolysis, we analyzed the reaction products by HPLC and found a greater abundance of pNP, indicating substantial monoesterase activity, i.e. pNPP hydrolysis (Fig. 7B). The ratio of pNPP:pNP implied that GP12 did not catalyze diester and monoester hydrolysis consecutively, which would have resulted in immediate conversion of the pNPP produced. This was verified by monitoring the course of P i release during bis-pNPP turnover. P i generation was observed at a significantly lower rate than pNP (Fig. 7C), providing strong evidence for catalysis of non-consecutive hydrolytic reactions by GP12 with phosphodiester-containing substrates.

Discussion
WTAs make up a substantial proportion of the cell wall in Gram-positive bacteria, where they adopt diverse functional roles. In B. subtilis, WTA degradation is thought to be part of the phosphate starvation response, but may also be important for cell wall recycling. An understanding of this process has been hampered by the absence of known enzymes possessing this activity, and lim-ited, in previous investigations, to impure enzyme preparations and/or uncharacterized WTA substrates. Here, we have initiated the clarification of this facet of WTA biochemistry by using synthetic WTA analogs to investigate teichoicase activity by the GP12 protein from bacteriophage 29. To our knowledge, this represents the first detailed study of such activity for a known enzyme, with chemically defined WTA substrates.
Given the large amount of WTA in the B. subtilis cell wall, it may not be surprising to find teichoicase activity of bacteriophage origin. An ability to efficiently degrade the glycosylated WTA encountered at the extracellular surface undoubtedly aids in overcoming the impediment to penetration posed by the cell wall. Still, such activity has only been reported for 29, although WTAs are well known bacteriophage cell-surface receptors (38). It is conceivable that other bacteriophages harbor teichoicase activity and indeed, a BLAST query on GP12 returns a number of putative homologs from Bacillus and Staphylococcus bacteriophages sharing the intramolecular chaperone and pectin lyase domains in common with GP12. Interestingly, these domains are also putatively identified in the product from the B. subtilis gene of unknown function, yobO. This gene product lacks significant sequence similarity to hypothetical B. subtilis teichoicases such as the phosphate starvation proteins PhoD and GlpQ (39), which invokes the possibility of multiple teichoicases encoded by the B. subtilis genome.  Whether these proteins exhibit teichoicase activity is the subject of ongoing work in our laboratory. That WTA glycosylation was shown to promote more efficient polymer degradation by GP12 also seems to coincide with the presumed biological function of this protein. In all likelihood, this was primarily due to the different modes that were discerned for the action of GP12 against non-glycosylated and glycosylated WTA. Glycosylation unexpectedly appeared to exert an effect on the charge properties of WTA, evidenced by chromatographic profiles (anion exchange) showing the elution of glycosylated analogs in much lower NaCl concentrations than non-glycosylated analogs with equivalent phosphate content. Comparable observations have been reported from polyacrylamide gel electrophoresis of biological WTAs, where D-alanyl modifications can be ruled out as the cause as they are known to be lost during the WTA extraction process (40). Therefore, glycosylation may counteract potentially unfavorable interactions with the highly negatively charged WTA polymer that could hinder productive associations with GP12. As well, degradation of non-glycosylated WTA gave rise to diverse products that supplied multiple additional substrates for GP12. Hence, as the reaction progresses, there may be increasing competition for the formation of enzyme complexes with the full-length (non-glycosylated) substrate, further contributing to a reduced rate of degradation. One can imagine testing this with WTA substrates of different polymer lengths and glycosylation states, although such substrates are difficult to create with current methods.
Arguably, the product distribution resulting from degradation of the glycosylated analog matches the expectations for an enzyme that acts processively on a polymeric substrate (41)(42)(43)(44). Moreover, the GP12 trimer has the capacity to accommodate multiple polymeric substrates (27), a property associated with processively acting enzymes (45). Even so, taking into account that GP12 catalyzes two distinct hydrolytic reactions, it would seem that non-processive polymer degradation is a likely mode of action for this enzyme.
The catalysis of two hydrolytic reactions by GP12 is reminiscent of certain binuclear metallophosphatases that utilize a bimetallated active site to catalyze the hydrolysis of both phosphate ester bonds in a diester substrate. Non-consecutive catalysis of these hydrolytic reactions has been shown for the promiscuous glycerophosphodiesterase GdpQ from Enterobacter aerogenes (46 -48), bacteriophage phosphatase (49), and a cyclic phosphatase dihydrolase from Eggerthella lenta (50). Alternatively, purple acid phosphatases from pig and red kidney bean (51), as well as CthPnkp from Clostridium thermocellum (52), are binuclear metallophosphatases that reportedly catalyze consecutive phosphate ester hydrolyzes; the second hydrolytic reaction appears to occur without prior enzyme release of the monoester product from the first reaction. The hallmark of this mechanism is the production of P i at roughly half the rate of the alcohol products from hydrolysis. Accordingly, the rates of P i and pNP production during the reaction of GP12 with bis-pNPP reflect discontinuous release of P i . The distinction between consecutive versus non-consecutive phosphate ester hydrolysis with WTA substrates could not be easily gleaned due to the difficulty in resolving monoester and alcohol products from complex degradation product mixtures. However, considering the starting concentrations of diester linkages and extent of GP12-catalyzed depolymerization under the experimental conditions, diester hydrolysis appeared to greatly exceed P i release. We surmise that GP12 catalyzes non-consecutive hydrolytic reactions with WTA substrates.
It is implicit from the GP12 crystal structure that the two hydrolytic reactions occur at the same catalytic center (27), although different amino acid residues may be important for diesterase and monoesterase activities (52,53). In support of this, it was observed that as diester hydrolysis slowed during the bis-pNPP reaction (due to product inhibition by pNP; not shown), the rate of P i release was unaffected (see Fig. 7C). Interestingly, GP12 did not catalyze P i release from glycerol phosphate, but was active on pNPP, a molecule that bears some structural resemblance to the ligand co-crystallized with GP12 (N-cyclohexyl-2-aminoethanesulfonic acid) (27). In view of this, the regions flanking the phosphate group appear relevant to the specificity of GP12 hydrolysis. This notion is exemplified by the aforementioned purple acid phosphatases, for which hydrolytic activity was shown to be affected by such factors as the steric accessibility of the substrate and the pK a of the potential leaving group (51,54). Our findings suggest that GP12 has an increased propensity toward the hydrolysis of phosphodiester bonds flanked by bulky substituents (e.g. glucose-glycerol, nitrophenol), and these considerations could have implications for the activity of GP12 in a biological setting, where WTA substrates possess an unspecified organization of ␣-glucose and D-alanine modifications.
In summary, we have conducted the first detailed biochemical investigation of a purified WTA degrading enzyme, demonstrating GP12 to be a glycerophosphodiesterase that exercises diesterase and monoesterase activities in its degradation of WTA polymers. This dual capability makes GP12 exceptional among glycerophosphodiesterases and offers an intriguing potential paradigm for WTA degrading enzymes by highlighting a mechanism for phosphate liberation from WTA. The influence of glycosylation on the efficiency, specificity, and modality of polymer degradation was an important discovery because the dispersal of glycosylation sites and other modifications throughout the biological polymer remains unclear. GP12 and related enzymes may therefore find utility as tools to investigate structural modifications to WTAs. We hope that the insight into the enzymology of WTA degradation provided by this current work will aid the characterization of novel teichoicases, and find further application in elucidating the physiological relevance of this activity.