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, Phe⁷¹ and Met¹⁷⁶ in the oxyanion hole and Arg²⁹⁴ 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 α/β 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.

Keywords: catalysis; crystal structure; new HTA subfamily; serine esterase; substrate recognition.

FIG 1

Multiple-sequence alignment of Est22 with four bona fide acetyltransferases from the HTA family. Secondary structures of Est22 are shown above the alignment using ESPript. Helices are indicated by springs, strands by arrows, turns by TT letters, and 3₁₀ helices by η letters. Identical residues are shown in white on a black background, and similar residues are in bold black. Black squares indicate residues forming the catalytic triad. Stars indicate residues conserved in helix αD4 of acetyltransferases but variable in the corresponding helix (αLF) of Est22. Residues spatially near the active sites and conserved in acetyltransferases but variable in Est22 are marked by circles.

FIG 2

Phylogenetic analysis of Est22 and other members of the HTA family. The tree was constructed with the neighbor-joining method with a JTT-matrix-based model using 167 amino acid positions. Bootstrap analysis of 1,000 replicates was conducted, and values above 50% are shown. Esterases from family V were used as an outgroup. Sequences with crystal structures are indicated by triangles. Enzyme activities tested in this study are indicated by circles.

FIG 3

Biochemical characterization of esterase Est22. (A) Analysis of the hydrolytic activities of Est22 and a typical HTA (HiHTA from H. influenzae) against pNPC4. (B) Analysis of the transferase activities of Est22 and HiHTA against acetyl-CoA and Hse. For panels A and B, all reactions were conducted at both 30°C and 60°C, and graphs were drawn with the highest values. (C) Substrate specificity of Est22. (D) Effect of temperature on the activity of Est22. (E) Effect of pH on the activity of Est22. (F) Effect of NaCl on the activity of Est22. The graphs show data from triplicate experiments (mean ± standard deviation [SD]).

FIG 4

Overall structure of Est22. (A) Structure of an Est22 dimer. Two monomers are colored gray and cyan. (B) Gel filtration analysis of Est22 and markers. The three protein size markers are ovalbumin (43 kDa), conalbumin (75 kDa), and the reported protein E40 (136.8 kDa) (23). (C) Structural comparison of Est22 (cyan), HiHTA (magenta), and MekB (pink). (D) Structure of monomeric Est22. The α helices in the helical bundle domain are colored light orange, and α helices and β strands in the α/β hydrolase domain are colored cyan and magenta, respectively. The catalytic triad is shown as yellow sticks.

FIG 5

Analyses of the key amino acid residues in Est22 for substrate binding and catalysis. (A) Electrostatic surface of the L374D mutant modeled with pNPC4. The positively charged regions are shown in blue and the negatively charged regions in red. The substrate pNPC4 is shown as orange sticks. (B) Detailed structure of the mutant L374D modeled with pNPC4. Residues involved in the binding and catalysis of pNPC4 are shown as cyan sticks, and the substrate pNPC4 is shown as orange sticks. Interactions between L374D residues and pNPC4 within hydrogen-bond distance are shown as dashed lines. (C) Activities of Est22 mutants. The activity of wild-type Est22 was defined as 100%. (D) CD spectra of Est22 and its mutants.

FIG 6

Front view (upper) and side view (lower) of the electrostatic surfaces of Est22, DAC-AT, and MekB. The positively charged regions are shown in blue and the negatively charged regions in red. In the upper panels, CoA is shown as green sticks in Est22 and MekB as bound in the DAC-AT-CoA complex, and the entrances of the catalytic cavities are marked with red dashed circles. In the lower panels, the catalytic cavities of Est22, DAC-AT, and MekB are marked with blue dashed circles, conserved regions in the catalytic cavities are colored dark red, and variable regions are colored white.

FIG 7

Differences in the catalytic cavities among three HTA enzymes, i.e., Est22, DAC-AT, and MekB. (A) Potential CoA-binding residues in DAC-AT (gray) and the corresponding residues in Est22 (cyan). CoA is drawn as green sticks in Est22 as bound in the DAC-AT-CoA complex. (B) Superposition of active site regions of Est22 and the modeled HiHTA-Hse complex. Residues of Est22 (cyan) corresponding to the key Hse-binding residues Arg²¹² and Asp³³⁸ of HiHTA (magenta) are marked with red circles. (C) Superposition of active site regions of the modeled L374D-pNPC4 complex and HiHTA. The Arg²⁹⁴ residue of the L374D mutant, which plays a dominant role in binding pNPC4, is marked with a red circle. Residues from the L374D mutant are colored cyan and residues from HiHTA magenta. (D) Residues lining the wall of the catalytic cavity of the L374D mutant (cyan). (E) Residues in the catalytic cavity of MekB (pink). pNPC4 is drawn as orange sticks in MekB as bound in the modeled L374D-pNPC4 complex. (F) Superposition of active site regions of the modeled L374D-pNPC4 complex and MekB. Residues from the L374D mutant are colored cyan and residues from MekB pink.