A Novel Subfamily Esterase with a Homoserine Transacetylase-like Fold but No Transferase Activity

Abstract

Microbial esterases play important roles in deep-sea organic carbon degradation and cycling. Although they have similar catalytic triads and oxyanion holes, esterases are hydrolases and homoserine transacetylases (HTAs) are transferases. Because two HTA homologs were identified as acetyl esterases, the HTA family was recently divided into the bona fide acetyltransferase subfamily and the acetyl esterase subfamily. Here, we identified and characterized a novel HTA-like esterase, Est22, from a deep-sea sedimentary metagenomic library. Est22 could efficiently hydrolyze esters with acyl lengths of up to six carbon atoms but had no transacetylase activity, which is different from HTAs and HTA-like acetyl esterases. Phylogenetic analysis also showed that Est22 and its homologs form a separate branch of the HTA family. We solved the structures of Est22 and its L374D mutant and modeled the structure of the L374D mutant with p-nitrophenyl butyrate. Based on structural, mutational, and biochemical analyses, Phe 71 and Met 176 in the oxyanion hole and Arg 294 were revealed to be the key substrate-binding residues. A detailed structural comparison indicated that differences in their catalytic tunnels lead to the different substrate specificities of Est22 and the other two HTA subfamilies. Biochemical and sequence analyses suggested that Est22 homologs may have the same substrate recognition and catalysis mechanisms as Est22. Due to the significant differences in sequences, structures, and substrate specificities between Est22 (and its homologs) and the other two HTA subfamilies, we suggest that Est22 and its homologs represent a new subfamily in the HTA family.IMPORTANCE Microbial esterases play important roles in the turnover of organic carbon in the deep sea. Esterases and HTAs represent two groups of ␣/␤ hydrolases. Esterases catalyze the hydrolysis of simple esters and are widely used in the pharmaceutical and agrochemical industries, while HTAs catalyze the transfer of an acetyl group from acetyl-coenzyme A (CoA) to homoserine and are essential for microbial growth. Here, we report on a novel HTA-like esterase, Est22, from a deep-sea sediment. Because of the significant differences in sequences, structures, and substrate specificities of HTAs and HTA-like acetyl esterases, Est22 and its homologs represent a new subfamily in the HTA family. This study offers new knowledge regarding marine esterases.

Excellent Degradation Performance of a Versatile Phthalic Acid Esters-Degrading Bacterium and Catalytic Mechanism of Monoalkyl Phthalate Hydrolase

“…The MAPs assumed a similar binding pattern for all complexes ( Figure 6 C–F). The catalytic triad S125-H291-D259, the key site of hydrolase/esterase family, formed a hydrogen bond network, the similar state that was observed in other hydrolases [ 43 , 44 ]. Additionally, the hydroxyl of S125 formed hydrogen bonds with the oxygen atoms of the ester bond, and would initial nucleophilic attack towards carbon atom of ester bond.…”

Metagenomic screening for lipolytic genes reveals an ecology-clustered distribution pattern

“…Patatin-like-proteins and tannases (designated as P and T, respectively) were shaded in yellow. Other recent reported lipolytic families were shaded in magenta: 1, Est22 (Li et al 2017); 2, EstL28 (Seo et al 2014); 3, Rv0045c (Guo et al 2010); 4, EstGX1 (Jiménez et al 2012); 5, EstLiu (Rahman et al 2016); 6, EstY (Wu and Sun 2009); 7; EstGS (Nacke et al 2011); 8, EM3L4 (Lee et al 2011); 9, FLS18 (Hu et al 2010b); 10, Est903 (Jia et al 2019); 11, EstJ (Choi et al 2013); 12, PE10 (Jiang et al 2012); 13, Est12 (Wu et al 2013); 14, EstDZ2 (Zarafeta et al 2016); 15, Est9x (Jeon et al 2009); 16, Lip10 (Guo et al 2016); 17, EstGH (Nacke et al 2011); 18, EML1 (Jeon et al 2009); 19, FnL (Yu et al 2010); 20, EstP2K (Ouyang et al 2013); 21, LipA (Couto et al 2010); 22, LipSM54 (Li et al 2016); 23, MtEst45 (Lee 2016); 24, LipT (Chow et al 2012); 25, EstSt7 (Wei et al 2013); 26, Rlip1 (Liu et al 2009); 27, EstA (Chu et al 2008); 28, FLS12 (Hu et al 2010b); 29, lp_3505 (Esteban-Torres et al 2014). Outer ring: substrate specificity of corresponding clones towards different carbon chain length (C4 – C14) of triglycerides.…”

“…A recent update of this system resulted in addition of 11 families (IX to XIX) (Kovacic et al 2019). Besides the nineteen families, there are claims of novel families, such as Est22 (Li et al 2017), Est9X(Jeon et al 2009), LipSM54 (Li et al 2016) and EstDZ2 (Zarafeta et al 2016). To avoid an artificial inflation of the number of families, these novel families were mostly excluded during classification of newly identified lipolytic enzymes in previous studies (Hitch and Clavel 2019).…”

A De Novo Designed Esterase with p-Nitrophenyl Acetate Hydrolysis Activity

“…The backbone oxygen anion hole is found in almost all proteases and many esterases. In general, the protein backbone is more rigid than the side chain [ 26 , 27 , 28 , 29 ]. Therefore, when screening the designed results, it may be preferable to choose a design that uses a backbone NH group to form an oxygen anion hole.…”