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  • Since the dideoxy analogues and


    Since the 1,2-dideoxy analogues, and , served as substrates for TgTCEA and TgTCEB, respectively, with the higher affinity (lower ) and lower turnover number (lower ) than their authentic substrates, PosA and PosB (such trend was more evident on analogue for TgTCEB than on analog for TgTCEA), an intriguing question was raised whether and analogues behave as efficient inhibitors of TgTCEs. We then measured the TgTCE activity (at 4 mM PosA or PosB) in the presence of dideoxy analogues (at 4 or 10 mM), but no remarkable decrease in the enzyme activity was observed; the rate of Pa formation was equal to the sum of those from Pos and the analogue (data not shown). Currently, the synthesis of potent Pos analogue type inhibitors of TgTCEs, , phosphonate esters, ketomethylene or dehydromethylene isosters with a 1,2-dideoxy-type alcohol moiety, which are expected to bind more efficiently to TgTCEs, is underway in our monohydrochloride synthesis laboratory. Stability of the synthetic Pos analogues under the enzyme reaction conditions (in 50 mM potassium phosphate buffer, monohydrochloride synthesis 6.5) was assessed by determining their half-lives (), as shown in . The values for PosA, , , and were 42, 66, 41, and 52 h, respectively, and those for PosB, , , and were 6.3, 9.7, 6.1, and 7.6 h, respectively. The results demonstrate that the compounds having the PosA-type 6-acyl group are more stable than those having the PosB-type 6-acyl group, but no marked improvement of the stability was observed by changing alcohol units. Interestingly, the ratios of values compared to the natural substrates were almost the same between the A-type and B-type analogues having the same alcohol units. That is, the ratios for /PosA, /PosA, and /PosA were 1.6, 0.98, and 1.2, respectively, and those for /PosB, /PosB, and /PosB were 1.5, 0.97, and 1.2, respectively. These results indicate that the stabilities of Pos and their analogues depend not only on the structure of acyl unit, but also on the structure of alcohol unit. TgTCE belongs to the carboxylesterase family in the α/β-hydrolase fold superfamily, and specifically catalyzes intramolecular transesterification, but not hydrolysis. This non-ester-hydrolyzing carboxylesterase is an example of an enzyme with catalytic properties that are unpredictable from its primary structure. The fact that TgTCEA and TgTCEB did not form the hydrolytic product PaA- and PaB-acids from any Pos analogues supports the reaction mechanism we proposed previously: TgTCE has rigid mechanism for an intramolecular nucleophilic attack by a terminal hydroxyl group of the acyl unit of Pos, but not by water, to the acyl-enzyme complex, resulting in the formation of the five-membered ring of Pa. Based on the kinetic analysis, it was suggested that TgTCEA has more flexible substrate recognition than TgTCEB with respect to the alcohol moiety; the changes in the and values of TgTCEA toward A-type analogues relative to those toward PosA were moderate, whereas values of TgTCEB toward B-type analogues and value toward markedly decreased relative to those toward PosB. To clarify whether such features of each of TgTCEA and TgTCEB are shared between the isozymes (TgTCEA isozymes from petals [used in this study] and bulbs,, ; TgTCEB isozymes from pollen grains [used in this study], roots and leaves), comparative kinetic analysis toward the various substrate analogues synthesized in this study should be performed in due course. Such analyses of substrate recognition will give clues not only about the functional specialization of TgTCEA and TgTCEB, which diverged from a common ancestral enzyme, but also about the differentiation of unique non-ester-hydrolyzing carboxylesterase from a canonical ester-hydrolyzing carboxylesterase.
    Introduction Highly conserved formylglycine generating enzyme (FGE) analogs exist in organisms, from bacteria to even humans, for post-translational modification of proteins [1]. Since their discovery, researchers have implemented FGE as a protein engineering tool for the post-translational formation of unique aldehyde-bearing formylglycine amino acids to serve as functional sites for bio-orthogonal modification [2], surface immobilization [3], [4], or a range of other protein labeling applications [5]. A common peptide substrate recognized by many forms of FGE is a short motif having the generic amino acid pattern XCXPXRX [1], [2], [6], [7]. Within this motif, it is the sulfhydryl group of the leading cysteine that is oxidized by FGE for formation of an aldehyde on formylglycine [8]. The use of this conversion for creating aldehyde tagged proteins has been widely implemented in the protein engineering community either in vitro by adding FGE after expression of the protein of interest or alternatively in vivo by co-expression of FGE on an inducible expression vector [2], [9]. Such reactions are typically carried out using the FGE of Mycobacterium tuberculosis in the range of 30–37°C [2], [3], [10], [11], though the choice of protein to be tagged may be limited to those which are stable for extended periods of time at these temperatures. Native sulfatase sequences, such as the CTPSR motif of arylsulfatase A, have often been used as the substrate tag despite the ability of the M. tuberculosis FGE to accept substitutions in the greater XCXPXRX motif [8].