Structural insights into the catalytic mechanism of lovastatin hydrolase

Abstract

The lovastatin hydrolase PcEST from the fungus Penicillium chrysogenum exhibits enormous potential for industrial-scale applications in single-step production of monacolin J, the key precursor for synthesis of the cholesterol-lowering drug simvastatin. This enzyme specifically and efficiently catalyzes the conversion of lovastatin to monacolin J but cannot hydrolyze simvastatin. Understanding the catalytic mechanism and the structure–function relationship of PcEST is therefore important for further lovastatin hydrolase screening, engineering, and commercial applications. Here, we solved four X-ray crystal structures, including apo PcEST (2.3 Å), PcEST in complex with monacolin J (2.48 Å), PcEST complexed with the substrate analog simvastatin (2.4 Å), and an inactivated PcEST variant (S57A) with the lovastatin substrate (2.3 Å). Structure-based biochemical analyses and mutagenesis assays revealed that the Ser57 (nucleophile)–Tyr170 (general base)–Lys60 (general acid) catalytic triad, the hydrogen-bond network (Trp344 and Tyr127) around the active site, and the specific substrate-binding tunnel together determine efficient and specific lovastatin hydrolysis by PcEST. Moreover, steric effects on nucleophilic attack caused by the 2′,2-dimethybutyryl group of simvastatin resulted in no activity of PcEST on simvastatin. On the basis of structural comparisons, we propose several indicators to define lovastatin esterases. Furthermore, using structure-guided enzyme engineering, we developed a PcEST variant, D106A, having improved solubility and thermostability, suggesting a promising application of this variant in industrial processes. To our knowledge, this is the first report describing the mechanism and structure–function relationship of lovastatin hydrolase and providing insights that may guide rapid screening and engineering of additional lovastatin esterase variants.

Crystal structure of a family VIII β‐lactamase fold hydrolase reveals the molecular mechanism for its broad substrate scope

“…Figure 5 gives a detailed picture of the residues delineating the active site in the wild‐type and EH 7 ‐M‐4NHP and EH 7 ‐O‐4NHP complexes. Structural comparison (not shown) of our complex with other reported EH 7 family enzymes complexed with substrates, such as the Est‐Y29/( S )‐ketoprofen (PDB Code 5ZWR ) [ 24 ] or the PcEst S57A/lovastatin (PDB Code 6KJD ) [ 9 ] complexes, ascribes the wide S1 to the acyl‐binding site of EH 7 and its homologues. As observed in Figs 5C,D and S1 site is an open cavity where apart from Tyr145 hydrogen‐bonded to Lys79, the side chains of Tyr75, Phe147 and Met366 create a hydrophobic environment for the proximal moiety of the acyl site, the remaining is exposed to the solvent due to its shorter and retracted R1.…”

“…The catalytic triad of EH 7 is formed by Ser76, Lys79 and Tyr192. Although the role of Tyr and Lys has been a matter of controversy, a recent study has given experimental evidence to propose that Tyr might act as a general base, whereas Lys could be the general acid aiding the positioning and polarization state of Tyr [ 9 ]. Nucleophilic serine and lysine are found in the S‐X‐X‐K motif, located at the beginning of α2, while conserved Tyr192 is located before α6.…”

“…Finally, the third conserved motif, which is part of the other wall of the catalytic cavity, is the K‐T‐G box. Family VIII is further subdivided into 3 subfamilies, VIII.1, VIII.2 and VIII.3, according to the conservation or absence of the previous motifs [ 8 ] [ 9 ]. Subfamily VIII.2 has a fourth conserved motif, G‐X‐S‐X‐G, that is common to family IV, in which the nucleophilic serine is included [ 10 ] (Table 1 ).…”