Novel Aldo-Keto Reductases for the Biocatalytic Conversion of 3-Hydroxybutanal to 1,3-Butanediol: Structural and Biochemical Studies

Abstract

The nonnatural alcohol 1,3-butanediol (1,3-BDO) is a valuable building block for the synthesis of various polymers. One of the potential pathways for the biosynthesis of 1,3-BDO includes the biotransformation of acetaldehyde to 1,3-BDO via 3-hydroxybutanal (3-HB) using aldolases and aldo-keto reductases (AKRs). This pathway requires an AKR selective for 3-HB, but inactive toward acetaldehyde, so it can be used for one-pot synthesis. In this work, we screened more than 20 purified uncharacterized AKRs for 3-HB reduction and identified 10 enzymes with significant activity and nine proteins with detectable activity. PA1127 from Pseudomonas aeruginosa showed the highest activity and was selected for comparative studies with STM2406 from Salmonella enterica serovar Typhimurium, for which we have determined the crystal structure. Both AKRs used NADPH as a cofactor, reduced a broad range of aldehydes, and showed low activities toward acetaldehyde. The crystal structures of STM2406 in complex with cacodylate or NADPH revealed the active site with bound molecules of a substrate mimic or cofactor. Site-directed mutagenesis of STM2406 and PA1127 identified the key residues important for the activity against 3-HB and aromatic aldehydes, which include the residues of the substrate-binding pocket and C-terminal loop. Our results revealed that the replacement of the STM2406 Asn65 by Met enhanced the activity and the affinity of this protein toward 3-HB, resulting in a 7-fold increase in k cat /K m . Our work provides further insights into the molecular mechanisms of the substrate selectivity of AKRs and for the rational design of these enzymes toward new substrates.IMPORTANCE In this study, we identified several aldo-keto reductases with significant activity in reducing 3-hydroxybutanal to 1,3-butanediol (1,3-BDO), an important commodity chemical. Biochemical and structural studies of these enzymes revealed the key catalytic and substrate-binding residues, including the two structural determinants necessary for high activity in the biosynthesis of 1,3-BDO. This work expands our understanding of the molecular mechanisms of the substrate selectivity of aldo-keto reductases and demonstrates the potential for protein engineering of these enzymes for applications in the biocatalytic production of 1,3-BDO and other valuable chemicals.KEYWORDS aldo-keto reductase, 1,3-butanediol, crystal structure, site-directed mutagenesis, biocatalysis, biotechnology P resently, most of the large-volume chemicals and polymer building blocks used by the chemical industry are manufactured from nonrenewable feedstocks, such as oil and natural gas. Each year, over 80 million tons of industrial chemicals are produced

Discovery of a Cryptic Intermediate in Late Steps of Mithramycin Biosynthesis

“…The PEG is bound in the substrate binding pocket because of its proximity to the nicotinamide ring of NADP + and Tyr 57. The structurally analogous substrate binding pocket STM2406 (Supplemental Figure 17, panels B and C) was functionally validated by mutagenesis . The loops that were disordered in the apo‐ and NADP + ‐bound crystal structures were all ordered in the MtmW‐NADP + ‐PEG crystal structure.…”

Crystal Structure and Biophysical Analysis of Furfural-Detoxifying Aldehyde Reductase from Clostridium beijerinckii

“…Single-crystal X-ray structure. The protein was cocrystallized with NADPH and the structure solved by molecular replacement using PDB entry 5T79, which is the crystal structure for STM2406, an AKR from Salmonella enterica serovar Typhimurium of unknown physiological function but with a substrate profile similar to that of Cbei_3974 (21). The two proteins have 60.91% sequence identity and a root mean square deviation of 0.89.…”

“…Both this structure and STM2406 have an unusual N terminus consisting of a long loop and a ␤-hairpin. Most AKR structures, including the structure of Coptotermes gestroi AKR1 (another AKR known to reduce furfural), have a shorter N-terminal tail consisting of only the ␤-hairpin or, in the case of the AKR7 family, have no N-terminal tail (21,23,24). The function of this extra sequence is unclear.…”

“…Neighboring aspartate and lysine residues lower the pK a of tyrosine to enable it to function as an acid (12). In the close homologue STM2406, the catalytic tetrad consists of Tyr-66, Asp61, Lys-97, and His-138 (21). All these residues are conserved in Cbei_3974 (identical numbering).…”

“…The carbonyl oxygen of 4-pyridine carboxaldehyde is within hydrogen bonding distance of the exocyclic amide of NADPH and makes hydrophobic contacts with residues Asn-65, Trp-33, and Tyr-100. These residues are conserved in STM2406 and have been shown to be important for binding in that enzyme (21). Furfural docked into the active site in a similar location but with a different orientation, possibly due to its smaller size.…”