The three Mycobacterium tuberculosis antigen 85 isoforms have unique substrates and activities determined by non-active site regions

Abstract

The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro(216)-Phe(228) loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors.

Keywords: Antigen 85; Cell Wall; Enzyme Mechanism; Glycolipid; Molecular Dynamics; Mycolyl Transferase; Trehalose; Tuberculosis.

FIGURE 1.

Activities of antigen 85. Antigen 85 catalyzes a transesterification reaction between an acyl donor (typically TMM) and an acceptor that can be free trehalose, another molecule of TMM, or the terminal arabinofuranose residues covalently anchored to the cell wall heteropolymer. The three common substrates are shown at the top, and the products of the enzymatic reaction are shown below each. Because they are highly insoluble, both cell wall polymer and TDM are likely to be terminal sinks for mycolic acids.

SCHEME 1.

Synthesis of hexasaccharide acceptor Ara6. DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; TMSOTf, trimethylsilyl trifluoromethanesulfonate.

FIGURE 2.

Previously reported structures and sequence alignments of the Ag85s, showing the high homology between the Ag85A, -B, and -C isoforms. PDB entries 1F0N, 1DQY, and 1DQZ are shown. A, the difference in the position of helix α9 (apo-structure 1DQZ (blue) and diethylphosphate acylated 1DQY (cyan)), adjacent Pro²¹⁶–Phe²²⁸ loop (blue/cyan), and key amino acids from Ag85C (Trp¹⁵⁷, Trp¹⁵⁸, and Thr²³¹; yellow) and from Ag85A/B (Met¹⁵⁹, Phe²³², and Val²³³; green), highlighting the differences between Ag85C and Ag85A/B at a secondary site that has been found with bound trehalose (1F0N, orange carbon, red oxygen atoms) or octylthioglucose (not shown). The catalytic triad is highlighted in magenta. B, the movement of helix α9 in A results in a disruption of the hydrogen bonding of the catalytic triad (dotted lines). The hydrogen-bonded triad in Ag85C structure 1DQZ is shown in magenta, and the disrupted triad in 1DQY is shown in cyan. C, sequence alignment of Ag85A, -B, and -C indicates the highly conserved nature of the enzymes. The box indicates sequence corresponding to helix α9 and the Pro²¹⁶–Phe²²⁸ loop, and the catalytic triad is indicated with arrows. Stars indicate the amino acids highlighted in A. Cyan, homology with Ag85A; magenta, homology with or unique to Ag85B; white, amino acids that are unique to Ag85C. Sequences are shown without signal peptides, and sequence numbering has been made consistent with that used previously (39–41).

FIGURE 3.

Site-directed mutagenesis revealed that non-conserved amino acids in a secondary site of Ag85 control the relative rates of acyl transfer and may explain the previously observed differences in catalytic activity between Ag85A, -B, and -C. A–C, MS acyltransferase assays measuring the acyl transfer from TDH to trehalose (Tre) to generate trehalose monohexanoate (TMH). Selected amino acids were replaced with either the amino acids from the other Ag85 isoform (e.g. Ag85A/B residues in Ag85C) or alanine. A, Ag85C mutants; B, Ag85B mutants; C, Ag85A mutants. D and E, acyl transfer assays with the native trehalose monomycolate (¹⁴C-TMM) substrate in the presence or absence of trehalose or 6-azido-trehalose as competitive substrates demonstrates a previously unknown difference in substrate specificity between Ag85A, -B, and -C, where C exhibits increased mycolation of free sugars, and A and B are more selective for TMM mycolation even in the presence of high concentrations of competing trehalose (200 μm). D shows the Ag85 TMM assay in the absence or presence of trehalose. E, TMM assays are conducted in the presence of competitive substrate 6-azido-trehalose (200 μm), which cannot form TDM. Ag85C, again, shows increased mycolation rates of the free sugar, whereas Ag85A, even in the presence of high concentrations of competitive substrate, selectively forms TDM. TLCs were developed in 80:20:2 chloroform/methanol/water.

FIGURE 4.

Molecular dynamics simulations show that mutations at Thr²³¹/Val²³³ alter the helix flexibility and the hydrogen bonding of the catalytic triad. A, degree of motion for Ag85C (PDB entry 1DQZ) and mutants (W157M, W158G, L230F, and T231V/A) measured by RMSDs of C-α atoms from molecular dynamics trajectories. Thicker tubes correspond to a higher RMSD. Side chains for residues forming part of the catalytic triad are represented as sticks. B, mutations at the analogous position in Ag85A (M159W, G160W, L233F, and V233T/A) result in changes in flexibility at the base of helix 9 and adjacent loop (residues Pro²¹⁶–Phe²²⁸) or nearby helix α5. V233T mutation confers Ag85C-like helical flexibility.

FIGURE 5.

Differences in flexibility of the residues comprising the base of helix α9 and an adjacent loop and helix α5 correlate with changes in substrate specificity and disparity in catalytic activity. A and B, relative rates of acylation and hydrolysis were calculated as in Fig. 2. Mutations to Thr²³¹ in Ag85C (A) dramatically alter the enzyme acyltransferase and acyl hydrolase activity in the trehalose dihexanoate assay as do mutations to the corresponding position in Ag85A (Val²³³) (B). C, mutation of Ag85C Thr²³¹ to the bulkier Val, Leu, or Ile decreases the relative formation of 6-azido-TMM, suggesting that structural changes in helix α9 and nearby regions determine substrate specificity. D, mutation of Ag85A to the less bulky Ag85C-like residues V231T and V231A increases formation of 6-azido-TMM. The Ag85A M159W/G160W/F232L (WWL) mutant demonstrates decreased formation of TDM but does not cause increased mycolation of 6-azido-TMM. TLCs were developed in 80:20:2 chloroform/methanol/water.

FIGURE 6.

The Ag85 substrate specificity may extend toward fragments of the mycobacterial cell wall, suggesting that the observed differences in enzyme activity may have implications in the partitioning of exported mycolates between TDM and the arabinan. A, Ara5 is docked in the active site of Ag85A (PDB entry 1SFR), revealing an extended active site that accommodates the bulk of the larger sugar. Residues are colored as in Fig. 1A. B, structures of docked Ara5 and assayed Ara6 are shown. My, mycolate attachment site. C, Ag85 TMM assays in the presence of Ara6 (500 μm) and competing trehalose (200 μm) reveal the formation of a new lipid species (*). Ag85A is most efficient at producing this new species from TMM. The new lipid (*) was the only significant product from the transesterification from TMM-6-azide to Ara6. TLC was developed in 80:20:2 chloroform/methanol/water. D, Ag85A-catalyzed formation of the new lipid species (*) in the absence (i) and presence (ii) of Ara6. E, the same TLC shown in D developed a second time in 95:5 chloroform/methanol to separate the solvent front lipid species, as indicated by the arrow. F, H37Rv M. tuberculosis treated with EMB at 10 times the minimum inhibitory concentration shows the accumulation of a new lipid species (**). TLC was developed in 100:10:0.1 chloroform/methanol/water. SQ109, which blocks the export of TMM, abolishes the formation of this species (**). G, the new lipid (**) in the EMB-treated 24-h sample from F has an Rf comparable with that of the product of the Ag85-catalyzed mycolation of Ara6 (*). TLC was developed twice first in 80:20:2, chloroform/methanol/water and then in 95:5 chloroform/methanol. Mycolation of Ara6 could not be increased further with the addition of higher concentrations of trehalose.