The ability of Mycobacterium tuberculosis to derive lipids from the host, store them intracellularly, and then break them down into energy requires a battery of serine hydrolases. Serine hydrolases are a large, diverse enzyme family with functional roles in dormant, active, and reactivating mycobacterial cultures. To rapidly measure substrate-dependent shifts in mycobacterial serine hydrolase activity, we combined a robust mycobacterial growth system of nitrogen limitation and variable carbon availability with nimble in-gel fluorogenic enzyme measurements. Using this methodology, we rapidly analyzed a range of ester substrates, identified multiple hydrolases concurrently, observed functional enzyme shifts, and measured global substrate preferences. Within every growth condition, mycobacterial hydrolases displayed the full, dynamic range of upregulated, downregulated, and constitutively active hydrolases independent of the ester substrate. Increasing the alkyl chain length of the ester substrate also allowed visualization of distinct hydrolase activity likely corresponding with lipases most responsible for lipid breakdown. The most robust expression of hydrolase activity was observed under the highest stress growth conditions, reflecting the induction of multiple metabolic pathways scavenging for energy to survive under this high stress. The unique hydrolases present under these high-stress conditions could represent novel drug targets for combination treatment with current front-line therapeutics. Combining diverse fluorogenic esters with in-gel activity measurements provides a rapid, customizable, and sensitive detection method for mycobacterial serine hydrolase activity.

BACKGROUND: Sophorolipids (SLs) are a class of natural, biodegradable surfactants that found their way as ingredients for environment friendly cleaning products, cosmetics and nanotechnological applications. Large-scale production relies on fermentations using the yeast Starmerella bombicola that naturally produces high titers of SLs from renewable resources. The resulting product is typically an extracellular mixture of acidic and lactonic congeners. Previously, we identified an esterase, termed Starmerella bombicola lactone esterase (SBLE), believed to act as an extracellular reverse lactonase to directly use acidic SLs as substrate.
RESULTS: We here show based on newly available pure substrates, HPLC and mass spectrometric analysis, that the actual substrates of SBLE are in fact bola SLs, revealing that SBLE actually catalyzes an intramolecular transesterification reaction. Bola SLs contain a second sophorose attached to the fatty acyl group that acts as a leaving group during lactonization.
CONCLUSIONS: The biosynthetic function by which the Starmerella bombicola \'lactone esterase\' converts acidic SLs into lactonic SLs should be revised to a \'transesterase\' where bola SL are the true intermediate. This insights paves the way for alternative engineering strategies to develop designer surfactants.

The utilization of polyethylene terephthalate (PET) has caused significant and prolonged ecological repercussions. Enzymatic degradation is an environmentally friendly approach to addressing PET contamination. Hydrolysis of mono(2-hydroxyethyl) terephthalate (MHET), a competitively inhibited intermediate in PET degradation, is catalyzed by MHET degrading enzymes. Herein, we employed bioinformatic methods that combined with sequence and structural information to discover an MHET hydrolase, BurkMHETase. Enzymatic characterization showed that the enzyme was relatively stable at pH 7.5-10.0 and 30-45 ℃. The kinetic parameters kcat and Km on MHET were (24.2±0.5)/s and (1.8±0.2) μmol/L, respectively, which were similar to that of the well-known IsMHETase with higher substrate affinity. BurkMHETase coupled with PET degradation enzymes improved the degradation of PET films. Structural analysis and mutation experiments indicated that BurkMHETase may have evolved specific structural features to hydrolyze MHET. For MHET degrading enzymes, aromatic amino acids at position 495 and the synergistic interactions between active sites or distal amino acids appear to be required for MHET hydrolytic activity. Therefore, BurkMHETase may have substantial potential in a dual-enzyme PET degradation system while the bioinformatic methods can be used to broaden the scope of applicable MHETase enzymes.

The human malaria parasite Plasmodium falciparum requires exogenous fatty acids to support its growth during the pathogenic, asexual erythrocytic stage. Host serum lysophosphatidylcholine (LPC) is a significant fatty acid source, yet the metabolic processes responsible for the liberation of free fatty acids from exogenous LPC are unknown. Using an assay for LPC hydrolysis in P. falciparum-infected erythrocytes, we have identified small-molecule inhibitors of key in situ lysophospholipase activities. Competitive activity-based profiling and generation of a panel of single-to-quadruple knockout parasite lines revealed that two enzymes of the serine hydrolase superfamily, termed exported lipase (XL) 2 and exported lipase homolog (XLH) 4, constitute the dominant lysophospholipase activities in parasite-infected erythrocytes. The parasite ensures efficient exogenous LPC hydrolysis by directing these two enzymes to distinct locations: XL2 is exported to the erythrocyte, while XLH4 is retained within the parasite. While XL2 and XLH4 were individually dispensable with little effect on LPC hydrolysis in situ, loss of both enzymes resulted in a strong reduction in fatty acid scavenging from LPC, hyperproduction of phosphatidylcholine, and an enhanced sensitivity to LPC toxicity. Notably, growth of XL/XLH-deficient parasites was severely impaired when cultured in media containing LPC as the sole exogenous fatty acid source. Furthermore, when XL2 and XLH4 activities were ablated by genetic or pharmacologic means, parasites were unable to proliferate in human serum, a physiologically relevant fatty acid source, revealing the essentiality of LPC hydrolysis in the host environment and its potential as a target for anti-malarial therapy.

Human acyl protein thioesterases (APTs) catalyze the depalmitoylation of S-acylated proteins attached to the plasma membrane, facilitating reversible cycles of membrane anchoring and detachment. We previously showed that a bacterial APT homologue, FTT258 from the gram-negative pathogen Francisella tularensis, exists in equilibrium between a closed and open state based on the structural dynamics of a flexible loop overlapping its active site. Although the structural dynamics of this loop are not conserved in human APTs, the amino acid sequence of this loop is highly conserved, indicating essential but divergent functions for this loop in human APTs. Herein, we investigated the role of this loop in regulating the catalytic activity, ligand binding, and protein folding of human APT1, a depalmitoylase connected with cancer, immune, and neurological signaling. Using a combination of substitutional analysis with kinetic, structural, and biophysical characterization, we show that even in its divergent structural location in human APT1 that this loop still regulates the catalytic activity of APT1 through contributions to ligand binding and substrate positioning. We confirmed previously known roles for multiple residues (Phe72 and Ile74) in substrate binding and catalysis while adding new roles in substrate selectivity (Pro69), in catalytic stabilization (Asp73 and Ile75), and in transitioning between the membrane binding β-tongue and substrate-binding loops (Trp71). Even conservative substitution of this tryptophan (Trp71) fulcrum led to complete loss of catalytic activity, a 13°C decrease in total protein stability, and drastic drops in ligand affinity, indicating that the combination of the size, shape, and aromaticity of Trp71 are essential to the proper structure of APT1. Mixing buried hydrophobic surface area with contributions to an exposed secondary surface pocket, Trp71 represents a previously unidentified class of essential tryptophans within α/β hydrolase structure and a potential allosteric binding site within human APTs.

Staphylococcus aureus is a major human pathogen responsible for a wide range of systemic infections. Since its propensity to form biofilms in vivo poses formidable challenges for both detection and treatment, tools that can be used to specifically image S. aureus biofilms are highly valuable for clinical management. Here we describe the development of oxadiazolonebased activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologs in other bacteria, it is an ideal target for selective imaging of S. aureus infections. Using X-ray crystallography, direct cell labeling and mouse models of infection we demonstrate that oxadiazolone-based probes enable specific labeling of S. aureus bacteria through the direct covalent modification of the FphE active site serine. These results demonstrate the utility of the oxadizolone electrophile for activity-based probes (ABPs) and validate FphE as a target for development of imaging contrast agents for the rapid detection of S. aureus infections.

An abbreviated synthesis of the cell permeable fluorophosphonate-alkyne probe (FP-alkyne) for the broad assessment of serine hydrolase activity has been developed. While FP-alkyne has proven pivotal in numerous chemical biology studies access has relied on a lengthy preparation over nine steps. We have developed a four-step synthesis, starting from commercially available compounds, with three purification steps to provide a new expedited route allowing easy access to a useful tool compound for exploring serine hydrolases chemistry and biology. This route was used in our own studies to generate FP-alkyne which in turn was used to identify the enzyme responsible for Fatty Acid Esters of Hydroxy Fatty Acids (FAHFA) biosynthesis. The use of this route can enable the syntheses of new tool compounds in addition to improving accessibility to FP-alkyne.

Mycobacterium abscessus, an opportunistic pathogen responsible for pulmonary infections, contains genes predicted to encode two steroid catabolic pathways: a cholesterol catabolic pathway similar to that of Mycobacterium tuberculosis and a 4-androstenedione (4-AD) catabolic pathway. Consistent with this prediction, M. abscessus grew on both steroids. In contrast to M. tuberculosis, Rhodococcus jostii RHA1, and other Actinobacteria, the cholesterol and 4-AD catabolic gene clusters of the M. abscessus complex lack genes encoding HsaD, the meta-cleavage product (MCP) hydrolase. However, M. abscessus ATCC 19977 harbors two hsaD homologs elsewhere in its genome. Only one of the encoded enzymes detectably transformed steroid metabolites. Among tested substrates, HsaDMab and HsaDMtb of M. tuberculosis had highest substrate specificities for MCPs with partially degraded side chains thioesterified with coenzyme A (kcat/KM = 1.9 × 104 and 5.7 × 103 mM-1s-1, respectively). Consistent with a dual role in cholesterol and 4-AD catabolism, HsaDMab also transformed nonthioesterified substrates efficiently, and a ΔhsaD mutant of M. abscessus grew on neither steroid. Interestingly, both steroids prevented growth of the mutant on acetate. The ΔhsaD mutant of M. abscessus excreted cholesterol metabolites with a fully degraded side chain, while the corresponding RHA1 mutant excreted metabolites with partially degraded side chains. Finally, the ΔhsaD mutant was not viable in macrophages. Overall, our data establish that the cholesterol and 4-AD catabolic pathways of M. abscessus are unique in that they converge upstream of where this occurs in characterized steroid-catabolizing bacteria. The data further indicate that cholesterol is a substrate for intracellular bacteria and that cholesterol-dependent toxicity is not strictly dependent on coenzyme A sequestration.

The 2.6 Å crystal structure of the apo form of Hip1 (hydrolase important for pathogenesis) has been previously reported. However, very little is known about the active site architecture of this M. tuberculosis (Mtb), serine hydrolase drug target. To begin mapping the active site of Hip1, we cocrystallized Hip1 with the irreversible serine protease inhibitor, 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF). We chose AEBSF for cocrystallization with Hip1 since the similar inhibitor, phenylmethylsulfonyl fluoride (PMSF), interestingly exhibited no activity against Hip1. We obtained crystals that diffracted to 2.1 Å but to our bewilderment, we did not observe any electron density for the inhibitor in the omit map for the Hip1-AEBSF complex. Rather, in the active site, dehydroalanine (dAla) was found to occupy the expected position of the catalytic Ser228, thus yielding anhydrohip1. Here we present a comparative analysis of the crystal structures of anhydrohip1 and Hip1 and provide a mechanism for the conversion of the enzyme to the anhydro-form through reaction with AEBSF. With the aid of molecular docking, we propose an explanation for the differential inhibition of Hip1 by AEBSF and PMSF. We also present a preliminary definition of the S1 and S2 pockets of the protease\'s active site and propose a mechanism for a ligand-induced conformational change within the S2 pocket. Finally, we expand upon the previous demarcation of the putative lipid binding pocket in the α-domain of the enzyme. We believe that this detailed analysis of the structures of anhydrohip1 and Hip1 provides valuable information useful for the structure-based drug design of novel Hip1-directed Mtb therapeutics.

Tripeptidyl peptidase II (TPPII or TPP2) degrades N-terminal tripeptides from proteins and peptides. Studies in both humans and mice have shown that TPPII deficiency is linked to cellular immune-senescence, lifespan regulation and the aging process. However, the mechanism of how TPPII participates in these processes is less clear. In this study, we established a chemical probe-based assay and found that although the mRNA and protein levels of TPPII were not altered during senescence, its enzymatic activity was reduced in senescent human fibroblasts. We also showed that elevation of the levels of the serine protease inhibitor serpinB2 reduced TPPII activity in senescent cells. Moreover, suppression of TPPII led to elevation in the amount of lysosomal contents as in well as TPPI (TPP1) and β-galactosidase activities, suggesting that lysosome biogenesis is induced to compensate for the reduction of TPPII activity in senescent cells. Together, this study discloses a critical role of the serpinB2-TPPII signaling pathway in proteostasis during senescence. Since serpinB2 levels can be increased by a variety of cellular stresses, reduction of TPPII activity through activation of serpinB2 might represent a common pathway for cells to respond to different stress conditions. This article has an associated First Person interview with the first author of the paper.