A D-carbamoylase NiHyuC from Nitratireductor indicus was identified with high catalytic activity toward Ncarbamoyl-D-tryptophan (3a). To further enhance its efficiency, both random mutagenesis and structure-guided evolution were performed. Variant M4 (D187N/A200N/S207A/R211G) showed a 43-fold increase in catalytic efficiency (k cat /K m = 1135.0 min −1 mM −1 ) and a 21-fold reduction in K m value (0.4 mM) compared with WT. Crystal structures of beneficial variants were resolved to clarify the evolutionary changes underlying improvements to catalytic efficiency. Structure alignment with WT indicated that loop 200−207 may play an important role in modulating access to the substrate entrance tunnel. Furthermore, MD simulations of WT−3a and M4−3a interactions illustrated that M4−3a has a better angle for nucleophilic attack and more readily enters a prereaction state. Additional hydrogen bonds and hydrophobic interactions were observed in prereaction states of M4−3a compared with that of WT−3a, consistent with its decreased K m value. In a hydantoinase process, the complete conversion of 160 mM Lindolylmethylhydantoin was achieved by M4 in a 0.5 L reaction, with D-tryptophan yield of 99.3% and productivity of 64.9 g L −1 d −1 . This study reveals a key loop at the substrate tunnel of D-carbamoylase and provides an effective strategy for engineering Dcarbamoylase and other carbon−nitrogen hydrolase family enzymes.

Methylenedioxy bridges (MDBs) are architecturally important motifs in natural products and bioactive molecules. Cercosporin, a typical perylenequinone pigment, contains an unusual seven-membered MDB, which has versatile biological and photocatalytic activities. Although cercosporin has been isolated, characterized, and studied for several decades, its biosynthetic pathway, especially the formation of the seven-membered MDB, has remained unclear. Here, we show that the formation of the seven-membered MDB is catalyzed by Fe­(II)/α-ketoglutaric acid (α-KG)-dependent dioxygenase CTB9 in cercosporin biosynthesis. Moreover, crystal structures of CTB9 in complex with an α-KG analogue NOG (CTB9·Cu·NOG) and its substrate pre-cercosporin with NOG (CTB9·Cu·NOG·pre-cercosporin) were determined. These structures, together with site-directed mutagenesis studies and quantum mechanics calculations, help define the mechanism of the unique seven-membered MDB in cercosporin biosynthesis. In summary, these results provide molecular insights into other biosynthetic pathways of natural products containing MDBs.

Steric hindrance modification-based pocket reshaping is an effective approach for enzyme–substrate acceptance evolution. However, previous studies were limited to using the unidirectional acceptance trait of bulky substrates as a measure of fitness. In this endeavor, we conducted steric hindrance modification-based bidirectional pocket reshaping of Exiguobacterium sibiricum leucine dehydrogenase (EsLeuDH) for elucidating the differences in the molecular mechanism of pocket steric hindrance on the adaptability between the bulky and small substrates. A site-directed mutagenesis library generated based on the specifically chosen bidirectional mutagenesis sites and building blocks enabled the substrate specificity of EsLeuDH to be extended to both the small aliphatic 1a and bulky aromatic 1h; the catalytic efficiency toward 1b–g increased by 2.5–16.3-fold. Kinetic parameter determination revealed that the increased acceptance of the mutants toward small and bulky substrates was attributed to the decreased K m and enhanced k cat values, respectively. Structure-based computational analysis provided insights into the increased acceptance in both the steric hindrance strengthening and attenuating directions, which was attributed to the reshaped pocket with a favorable attack distance and an expanded catalytical binding space, respectively. Our study elucidates the mechanism difference of pocket steric hindrance of EsLeuDH on the adaptability of different types of substrates based on the implementation of bidirectional pocket reshaping, with potential applications in the divergent substrate acceptance evolution of amino acid dehydrogenase family members and other oxidoreductases with analogous substrate-binding pocket.

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A cercosporin-catalyzed photo-mediated oxidation for the rapid and efficient synthesis of kynurenine (Kyn) derivatives as well as Kyn-contained peptides under mild conditions was developed, exhibiting great functional group tolerance and...