*2.1. Cloning of the Full-Size Endo-*α*-(1*→*6)-*d*-Mannanase Gene from Bacillus circulans TN-31*

Using reverse genetics, Maruyama et al. identified *aman6* as the gene encoding the endo-α-(1→6)-D-mannanase from *B. circulans* TN-31 by screening a *Sau*3A1-digested genomic DNA library of this bacterium. The *aman6* gene was reported to encode a 589-amino acid-long protein with a 36 amino-acid signal peptide (GenBank accession number AB024331) [10]. Attempts to PCR-amplify *aman6* in our laboratory using primers to the reported nucleotide sequence were unsuccessful. The nucleotide BLAST analysis of *aman6* yielded a gene, *gymC10\_1685* from *Paenibacillus sp.* Y412MC10, with 88% DNA sequence identity to *aman6* on the first 1722-bp but a substantially larger coding sequence totaling 3249-bp instead of 1767 bp for *aman6*. Using primers specific to the 5 -end of *aman6* and to the downstream region of *gymC10\_1685*, a 3522 bp-long PCR product was amplified from *B. circulans TN-31 genomic DNA*. The fragment encompasses a 3255 bp-long open reading frame encoding a 1084 amino acid protein with a 33 amino acid signal peptide (Figure S1). The molecular weight of the mature protein (131 kDa) matches that of the endo-α-(1→6)-D-mannanase purified by Ballou and collaborators from the same bacterium [8]. We named the full-size endomannanase gene *emn*. The mature Emn protein consists of a glycosyl hydrolase family 76 (GH-76) catalytic domain from amino acids 36 to 360 followed by three consecutive family six carbohydrate-binding modules from residues 392 to 522, 534 to 657 and 670 to 793, and a domain of unknown function (DUF4959) from residues 811 to 902. The GH-76 CaZy family of glycosyl hydrolases consists of α-(1→6)-mannanases (EC # 3.2.1.101) and α-glucosidases (EC # 3.2.1.20), whereas family six carbohydrate-binding modules are non-catalytic domains thought to facilitate the binding of the catalytic GH domain to its substrate(s).

#### *2.2. Expression and Purification of the Glycosyl Hydrolase Domain of Emn*

The production of the mature Emn protein from a pET14b expression plasmid in *E. coli* BL21 pLysS proved to be toxic to the cells and failed to yield any detectable protein product. We were, however, able to produce, using the same expression system, a 43 kDa N-terminally His6-tagged recombinant protein encompassing the glycosyl hydrolase domain of Emn (hereafter referred to as GH-emn) corresponding to residues 36 to 400 of the full-size enzyme (Figure S1) and to purify it to

near-homogeneity using a combination of nickel affinity chromatography and gel filtration (Figure S2). The purified GH-emn protein was stored at –80 ◦C in 20 mM Tris pH 7.5 buffer containing 150 mM NaCl and 20% glycerol for over 24 months without any appreciable loss of activity.

### *2.3. Digestion of Synthetic Mannoside Substrates*

The purified GH domain of Emn was then tested for glycosyl hydrolase activity using two synthetic unbranched linear substrates consisting of octyl trimannoside (α*-*d*-*Man*p*-(1→6)-α-d-Man-(1→6)-α-d-Man- (1→octyl)) and octyl pentamannoside (α-d-Man*p*-(1→6)-α-d-Man-(1→6)-α-d-Man-(1→6)-α-d-Man- (1→6)-α-d-Man-(1→octyl)). These substrates are structurally similar to those originally used by Ballou and co-workers [8] to study the substrate specificity of the purified, native, full-size enzyme. Those used by Ballou were unsubstituted at the reducing end, whereas those we evaluated harbor an octyl aglycon at their reducing end to facilitate the identification of the hydrolytic products of the reaction and thus, exactly where the enzyme cleaves. Enzyme assays were carried out as described under Materials and Methods in liquid chromatography—mass spectrometry (LC/MS) sample vials at 50 ◦C for 16 h [8]—after which the reaction mixture was directly injected into the LC/MS instrument without further purification. Figure 1A–B shows the base peak chromatograms of octyl trimannoside and its GH-emn digestion products. The mass spectrum of the only major peak found for the substrate (peak (i)) showed a major [M–H] ion at *m*/*z* 615.29 as well as an ion at *m*/*z* 675.30 [M+HAc–H] and an ion at *m*/*z* 661.29 [M+HCOOH–H], all corresponding to the parent molecule, Man*p*3-(1→octyl) (Figure 1C). In contrast, the base peak chromatogram of the enzymatic products revealed the loss of peak (i) with the concomitant appearance of two new peaks, (ii) and (iii). The mass spectrum of peak (ii) showed a strong ion at *m*/*z* 377.08 [M+Cl–] corresponding to Man*p*<sup>2</sup> (Figure 1D), whereas peak (iii) yielded a signal at *m*/*z* 351.20 [M+HAc–H] corresponding to Man*p*-(1→octyl) (Figure 1E). These data show that the enzyme cleaves the trisaccharide at glycosidic bond of the middle Man*p* residue (labeled as M2 in Figure 1F) unit rather than the glycosidic bond of the Man*p* residue at either end of the trimannoside unit (labeled as M1 and M3 in Figure 1F).

**Figure 1.** LC/MS analysis of the GH-emn-digested synthetic octyl trimannoside. (**A**,**B**) Base peak chromatograms showing the elution profiles of the substrate (**A**) and products of the enzymatic reaction (**B**). (**C**) Mass spectrum of the undigested synthetic octyl trimannoside at *m*/*z* 615.28 [M-H] as well as formate and acetate adducts. (**D**,**E**) Mass spectra of the products of the reaction: mannobiose (peak (ii)) (**D**) with a free reducing end at *m*/*z* 377.08 [M+Cl–] and Man*p*1-octyl (peak (iii)) (**E**) at *m*/*z* 291.18 [M-H], 337.18 (M+formate) and 351.2 (M+acetate). (**F**) Cartoon showing the location of the mannosyl cleavage (thicker arrow) of the octyl trimannoside at residue M2. The red arrow indicates the absence of cleavage by the enzyme.

A similar hydrolytic pattern was obtained upon digestion of the octyl pentamannoside by GH-emn (Figure 2). In general, Man*p*1-4 as free reducing components were found as [M+Cl–] ions and Man*p*1, 2, 3, and 5-(1→octyl) as [M+HAc–H] ions. The extracted ion chromatograms for these ions were integrated to obtain areas used to calculate the data presented in Figure 2A,B. The amounts of these ions varied with the enzyme concentration used (Figure 2A,B). Importantly, no Man*p*4-(1→octyl) was detected with any amount of enzyme, as predicted from the data of Man*p*3-(1→octyl) (Figure 1). Complete digestion occurred at the two highest enzyme concentrations (50 and 100 μg/mL) yielding only Man*p*2-(1→octyl) and Man*p*1-(1→octyl) in a ratio of approximately 2:1 and the free mannosides Man*p*<sup>2</sup> and Man*p*<sup>1</sup> in a 0.9:1 ratio. Traces of Man*p*3-(1→octyl) (Figure 2A) along with oligomannosides Man*p*<sup>4</sup> and Man*p*<sup>3</sup> (Figure 2B) were only detected when less enzyme was used in the assay, suggesting that these products are further cleaved to form Man*p*1-(1→octyl), and free Man*p*<sup>2</sup> and Man*p*1. Collectively, these results indicate that GH-emn cleaves the octyl pentamannoside substrate at three different positions, as summarized in Figure 2C. At all three cleavage positions, the enzyme binds to three mannosyl residues and cuts between the middle Man and the reducing end Man. Consistently with that binding, the enzyme cannot further process Man*p*2-(1→octyl) or Man2, in agreement with previous studies from Dr. Ballou's laboratory [8].

**Figure 2.** Digestion of octyl pentamannoside with increasing concentrations of GH-emn. (**A**) Percentage area of products containing an octyl chain. (**B**) Molar percentage of oligomannosides with a free reducing end. (**C**) Cartoon showing the locations of the three possible mannosyl cleavage sites. The thicker arrow denotes the dominant cleavage site having three mannosyl residues at the non-reducing end and the dotted arrows indicate other, less favored cleavage sites. The further cleavage of the released Man*p*<sup>4</sup> and Man*p*<sup>3</sup> is not shown.

#### *2.4. Digestion of Phosphatidyl-Myo-Inositol-Mannosides*

The reducing end of LM and LAM consists of phosphatidyl-*myo*-inositol where the *myo*-inositol residue is mannosylated at positions C-2 and C-6. This lipid anchor may be esterified with up to four acyl chains [1]. Phosphatidyl-*myo*-inositol mannosides, also known as PIMs, may also exist as free glycolipids populating the inner and outer membranes of mycobacteria [11]. Triand tetra-acylated phosphatidyl-*myo*-inositol dimannosides (Ac1PIM2 and Ac2PIM2) and tri- and tetra-acylated phosphatidyl-*myo*-inositol hexamannosides (Ac1PIM6 and Ac2PIM6) are the most abundant form of PIMs found in the mycobacterial cell envelope [11]. As expected from the results above, GH-emn showed no activity on deacylated PIM2 (d-PIM2) which lacks α-(1→6)-linked mannosyl residues (Figure S3).

Since earlier work has established that purified, native, Emn lacks activity on yeast mannan in which the backbone is substituted with Man*p-*α-(1→3)- and α-(1→2)-linked residues [7,8], we sought to determine whether GH-emn could cleave the pentamannoside extending from the six-position of *myo*-inositol in deacylated Ac2PIM6 (d-PIM6). In this molecule, the first three mannosyl residues attached to *O*-6 of the inositol are linked via α-(1→6) glycosidic linkages and the last two mannosyl residues are present as the dimannoside α-Man*p*-(1→2)-α-Man*p* attached to position 2 of the third Man*p* from the inositol residue (Figure 3). PIM6, as received from BEI, is a mixture containing both PIM2 (shown above to not be a substrate for GH-emn) and PIM6 in equal amounts. Hence, we needed to focus our analysis on the released free mannosides rather than the inositol-containing components. GH-emn activity on intact d-PIM6 was analyzed by LC/MS in the negative ion mode. Upon enzymatic treatment, the amount of intact d-PIM6 (MS and structure shown in Figure 3D) decreased dramatically (Figure 3A,B). Concomitantly, the chromatogram and mass spectrum of the enzyme-treated sample were dominated by a peak at *m*/*z* 701.19 [M+Cl–] corresponding to the tetramannoside, D-Man*p*-(1→2)-α-D-Man*p*-(1→2)-α-D-Man*p*-(1→6)-α-D-Man*p*-(1→6) (Figure 3C,E). No ions corresponding to the mono-, di-, tri- or penta- mannosides were observed in the enzyme-treated sample. These results indicate that the GH-emn can tolerate an α-(1→2)-linked dimannoside substitution at the non-reducing terminus (M4 in Figure 3F) of the α-(1→6 trimannoside component (M4, M3, and M2) of d-PIM6.

**Figure 3.** LC/MS analysis of undigested and GM-emn-digested deacylated PIM6. (**A**,**D**) Extracted Ion Chromatogram (**A**), structure (**D**), and mass spectrum (**D**) of d-PIM6 (*m*/*z* 652.18 [M–2H]) before enzyme treatment. (B) Extracted ion chromatograms (EIC) of d-PIM6 (*m*/*z* 652.18 [M–2H]) after digestion with GH-emn. The EIC of *m*/*z* 701.19 [M+Cl–] shown in panel (**C**) corresponds to the dominant tetramannoside product of d-PIM6 after digestion with GH-emn. Its mass spectrum is shown in panel (**E**). (**F**) Cartoon showing the location of the mannosyl cleavage site of GH-emn to form Man4. The red arrows indicate the absence of cleavage by the enzyme.

#### *2.5. Analysis of Endomannanase-Digested LAM and LM*

Next, we tested the endomannanase activity of GM-emn on the acylated forms of LM and LAM from *M. smegmatis*. Complete digestion of these substrates after an overnight incubation with GH-emn was evident from the analysis of the products of the reaction by SDS-PAGE followed by silver staining (Figure 4). Enzymatic digestion of LM and LAM further resulted in a low molecular weight product migrating slightly faster than acylated PIM6 (Figure 4, lanes 3, 5 and 6), which we tentatively attribute to di- or tri-mannosylated forms of phosphatidyl-*myo*-inositol released from the reducing end of both lipoglycans.

**Figure 4.** Silver-stained SDS PAGE showing the GH-emn digestion products of native *M. smegmatis* LM and LAM. MWM: Molecular weight marker.

To gain further insight into the nature of the fragments released by GH-emn from deacylated *M. smegmatis* LM (d-LM), the products of the reaction were also analyzed by LC/MS. The LC/MS profile of undigested d-LM was dominated by doubly, triply and quadruply charged, long-chain, high-molecular weight (range of 2,700 to 6,800 Da) mannans containing 15 to 40 mannosyl residues (Figure 5A). The most abundant components appeared as triply charged series [M–3H] of ions at *m*/*z* 1406.77, 1460.79, 1514.81, 1568.82, 1622.84, 1676.86 and 1730.88 corresponding to LM molecules with 24 to 34 mannosyl residues. The less abundant quadruply-charged series [M–4H] corresponded to mannans with 27 to 40 mannosyl residues, while the least abundant doubly-charged series contained 24 to 33 mannosyl residues. Together, these results confirm that the mannan backbone of *M. smegmatis* LM is composed of up to 40 mannosyl residues. GH-emn digestion of this heterogeneous population of d-LM resulted

in two major ions at *m*/*z* 657.16 and 819.21, corresponding to the mass of d-PIM2 and d-PIM3 released from the reducing end (Figure 5B). Additionally, the presence of oligomannans (with free reducing ends) was deduced from the presence of singly, doubly and triply charged series of ions matching the mass of Man*p*<sup>2</sup> and higher oligomers with odd numbers of mannosyl residues from Man*p*<sup>3</sup> to Man*p*<sup>27</sup> (Figure 5B). These oligomannans result from the presence of *O*-2 mannosyl substitutions at various positions of the mannan backbone of LM, preventing GH-emn from cutting. Their detailed structure awaits further characterization.

**Figure 5.** Negative ion mass spectra of deacylated LM from *M. smegmatis* before and after digestion with GH-emn. (**A**) Mass spectrum of undigested d-LM. The spectrum shows the mass distribution of the mannan backbone of d-LM which contains up to 40 mannosyl residues (differing by the mass of one hexose). Inset: The isotopic pattern for quadruply-charged d-LM with 40 mannosyl residues indicates the sensitivity of the mass spectrometer for high-molecular weight compounds. (**B**) Mass spectrum of d-LM after digestion by GH-emn. The mass spectra for three different retention times (RT) are shown. At all retention times, the spectra are dominated by singly-charged PIM2 and PIM3. RT 2.6 and 2.8 min show the presence of ions for Man17 to Man27 [M-3H] and Man9 to Man17 [M-2H]. RT 3.0 min shows low-molecular weight oligomannosides (Man2 to Man7) as singly charged and doubly charged chlorine adducts.
