Development of an Enzyme Cascade System for the Synthesis of Enantiomerically Pure D-Amino Acids Utilizing Ancestral L-Amino Acid Oxidase.

Enantiomerically pure D-amino acids hold significant potential as precursors for synthesizing various fine chemicals, including peptide-based drugs and other pharmaceuticals. This study focuses on establishing an enzymatic cascade system capable of converting various L-amino acids into their D-isomers. The system integrates four enzymes: ancestral L-amino acid oxidase (AncLAAO-N4), D-amino acid dehydrogenase (DAADH), D-glucose dehydrogenase (GDH), and catalase. AncLAAO-N4 initiates the process by converting L-amino acids to corresponding keto acids, which are then stereo-selectively aminated to D-amino acids by DAADH using NADPH and NH4Cl. Concurrently, any generated H2O2 is decomposed into O2 and H2O by catalase, while GDH regenerates NADPH from D-glucose. Optimization of reaction conditions and substrate concentrations enabled the successful synthesis of five D-amino acids, including a D-Phe derivative, three D-Trp derivatives, and D-phenylglycine, all with high enantiopurity (>99 % ee) at a preparative scale (>100 mg). This system demonstrates a versatile approach for producing a diverse array of D-amino acids.

Engineering the Substrate Specificity of (S)-ß-Phenylalanine Adenylation Enzyme HitB.

Adenylation enzymes catalyze the selective incorporation of aminoacyl building blocks in the biosynthesis of nonribosomal peptides and related natural products. Although ß-amino acid units are one of the important aminoacyl building blocks in natural product biosynthesis, very little is known about the engineering of ß-amino acid adenylation enzymes. In this study, we engineered the substrate specificity of the (S)-ß-phenylalanine adenylation enzyme, HitB, involved in the biosynthesis of macrolactam polyketide hitachimycin. Based on the previously determined structure of HitB wild-type, we mutated Phe328 and Ser293, which are located near the meta and ortho position of the (S)-ß-phenylalanine moiety, respectively. As a result, the HitB F328V and F328L mutants efficiently activated meta-substituted (S)-ß-phenylalanine analogs, and the HitB T293G and T293S mutants efficiently activated ortho-substituted (S)-ß-phenylalanine analogs. Structural analysis of the HitB F328L and T293G mutants with the corresponding nonhydrolyzable intermediate analogs revealed an enlarged substrate binding pocket for (S)-ß-phenylalanine analogs, providing detailed insights into the structural basis for creating enzyme substrate promiscuity. Our findings may be useful for production of various ß-amino acid-containing natural product analogs.

Structural Basis of Transient Interactions of Acyltransferase VinK with the Loading Acyl Carrier Protein of the Vicenistatin Modular Polyketide Synthase.

Acyltransferase (AT) recognizes its cognate acyl carrier protein (ACP) for functional transfer of an acyl unit in polyketide biosynthesis. However, structural characterization of AT-ACP complexes is limited because of the weak and transient interactions between them. In the biosynthesis of macrolactam polyketide vicenistatin, the trans-acting loading AT VinK transfers a dipeptidyl unit from the stand-alone ACP VinL to the ACP domain (VinP1ACPL) of the loading module of modular polyketide synthase VinP1. Although the previously determined structure of the VinK-VinL complex clearly illustrates the VinL recognition mechanism of VinK, how VinK recognizes VinP1ACPL remains unclear. Here, the crystal structure of a covalent VinK-VinP1ACPL complex formed with a pantetheine-type cross-linking probe is reported at 3.0 Å resolution. The structure of the VinK-VinP1ACPL complex provides detailed insights into the transient interactions between VinK and VinP1ACPL. The importance of residues in the binding interface was confirmed by site-directed mutational analyses. The binding interface between VinK and VinP1ACPL is similar to that between VinK and VinL, although some of the interface residues are different. However, the ACP orientation and interaction mode observed in the VinK-VinP1ACPL complex are different from those observed in other AT-ACP complexes such as the disorazole trans-AT-ACP complex and cis-AT-ACP complexes of modular polyketide synthases. Thus, AT-ACP binding interface interactions are different in each type of AT-ACP pair.

Protein-Protein Recognition Involved in the Intermodular Transacylation Reaction in Modular Polyketide Synthase in the Biosynthesis of Vicenistatin.

The ketosynthase (KS) domain is a core domain found in modular polyketide synthases (PKSs). To maintain the polyketide biosynthetic fidelity, the KS domain must only accept an acyl group from the acyl carrier protein (ACP) domain of the immediate upstream module even when they are separated into different polypeptides. Although it was reported that both the docking domain-based interactions and KS-ACP compatibility are important for the interpolypeptide transacylation reaction in 6-deoxyerythronolide B synthase, it is not clear whether these findings are broadly applied to other modular PKSs. Herein, we describe the importance of protein-protein recognition in the intermodular transacylation between VinP1 module 3 and VinP2 module 4 in vicenistatin biosynthesis. We compared the transacylation activity and crosslinking efficiency of VinP2 KS4 against the cognate VinP1 ACP3 with the noncognate one. As a result, it appeared that VinP2 KS4 distinguishes the cognate ACP3 from other ACPs.

Functional and Structural Analyses of the Split-Dehydratase Domain in the Biosynthesis of Macrolactam Polyketide Cremimycin.

In the biosynthesis of the macrolactam antibiotic cremimycin, the 3-aminononanoic acid starter unit is formed via a non-2-enoyl acyl carrier protein thioester intermediate, which is presumed to be constructed by cis-acyltransferase (AT) polyketide synthases (PKSs) CmiP2, CmiP3, and CmiP4. While canonical cis-AT PKS modules are comprised of a single polypeptide, the PKS module formed by CmiP2 and CmiP3 is split within the dehydratase (DH) domain. Here, we report the enzymatic function and the structural features of this split-DH domain. In vitro analysis showed that the split-DH domain catalyzes the dehydration reaction of (R)-3-hydroxynonanoyl N-acetylcysteamine thioester (SNAC) to form (E)-non-2-enoyl-SNAC, suggesting that the split-DH domain is catalytically active in cremimycin biosynthesis. In addition, structural analysis revealed that the CmiP2 and CmiP3 subunits of the split-DH domain form a tightly associated heterodimer through several hydrogen bonding and hydrophobic interactions, which are similar to those of canonical DH domains of other cis-AT PKSs. These results indicate that the split-DH domain has the same function and structure as common cis-AT PKS DH domains.

Structural Analysis of the Glycine Oxidase Homologue CmiS2 Reveals a Unique Substrate Recognition Mechanism for Formation of a ß-Amino Acid Starter Unit in Cremimycin Biosynthesis.

The flavin adenine dinucleotide-dependent oxidase CmiS2 catalyzes the oxidation of N-carboxymethyl-3-aminononanoic acid to produce a 3-aminononanoic acid starter unit for the biosynthesis of cremimycin, a macrolactam polyketide. Although the sequence of CmiS2 is similar with that of the well-characterized glycine oxidase ThiO, the chemical structure of the substrate of CmiS2 is different from that of ThiO substrate glycine. Here, we present the biochemical and structural characterization of CmiS2. Kinetic analysis revealed that CmiS2 has a strong preference for N-carboxymethyl-3-aminononanoic acid over other substrates such as N-carboxymethyl-3-aminobutanoic acid and glycine, suggesting that CmiS2 recognizes the nonanoic acid moiety of the substrate as well as the glycine moiety. We determined the crystal structure of CmiS2 in complex with a substrate analogue, namely, S-carboxymethyl-3-thiononanoic acid, which enabled the identification of key amino acid residues involved in substrate recognition. We discovered that Asn49, Arg243, and Arg334 interact with the carboxyl group of the nonanoic acid moiety, while Pro46, Leu52, and Ile335 recognize the alkyl chain of the nonanoic acid moiety via hydrophobic interaction. These residues are highly conserved in CmiS2 homologues involved in the biosynthesis of related macrolactam polyketides but are not conserved in glycine oxidases such as ThiO. These results suggest that CmiS2-type enzymes employ a distinct mechanism of substrate recognition for the synthesis of ß-amino acids.

Structural analysis of the dual-function thioesterase SAV606 unravels the mechanism of Michael addition of glycine to an α,ß-unsaturated thioester.

Thioesterases catalyze hydrolysis of acyl thioesters to release carboxylic acid or macrocyclization to produce the corresponding macrocycle in the biosynthesis of fatty acids, polyketides, or nonribosomal peptides. Recently, we reported that the thioesterase CmiS1 from Streptomyces sp. MJ635-86F5 catalyzes the Michael addition of glycine to an α,ß-unsaturated fatty acyl thioester followed by thioester hydrolysis in the biosynthesis of the macrolactam antibiotic cremimycin. However, the molecular mechanisms of CmiS1-catalyzed reactions are unclear. Here, we report on the functional and structural characterization of the CmiS1 homolog SAV606 from Streptomyces avermitilis MA-4680. In vitro analysis indicated that SAV606 catalyzes the Michael addition of glycine to crotonic acid thioester and subsequent hydrolysis yielding (R)-N-carboxymethyl-3-aminobutyric acid. We also determined the crystal structures of SAV606 both in ligand-free form at 2.4 Å resolution and in complex with (R)-N-carboxymethyl-3-aminobutyric acid at 2.0 Å resolution. We found that SAV606 adopts an α/ß hotdog fold and has an active site at the dimeric interface. Examining the complexed structure, we noted that the substrate-binding loop comprising Tyr-53-Asn-61 recognizes the glycine moiety of (R)-N-carboxymethyl-3-aminobutyric acid. Moreover, we found that SAV606 does not contain an acidic residue at the active site, which is distinct from canonical hotdog thioesterases. Site-directed mutagenesis experiments revealed that His-59 plays a crucial role in both the Michael addition and hydrolysis via a water molecule. These results allow us to propose the reaction mechanism of the SAV606-catalyzed Michael addition and thioester hydrolysis and provide new insight into the multiple functions of a thioesterase family enzyme.

Structural Basis of Amide-Forming Adenylation Enzyme VinM in Vicenistatin Biosynthesis.

Adenylation enzymes activate amino acid substrates to aminoacyl adenylates and generally transfer this moiety onto the thiol group of the phosphopantetheine arm of a carrier protein for the selective incorporation of aminoacyl building blocks in natural product biosynthesis. In contrast to the canonical thioester-forming adenylation enzymes, the amide-forming adenylation enzyme VinM transfers an l-alanyl group onto the amino group of the aminoacyl unit attached to the phosphopantetheine arm of the carrier protein VinL to generate dipeptidyl-VinL in vicenistatin biosynthesis. It is unclear how VinM distinguishes aminoacyl-VinL from VinL for amide bond formation. Herein we describe structural and biochemical analyses of VinM. We determined the crystal structure of VinM in complex with VinL using a designed pantetheine-type cross-linking probe. The VinM-VinL complex structure in combination with site-directed mutagenesis analysis revealed that the interactions with both the phosphopantetheine arm and VinL are critical for the amide-forming activity of VinM.

Structure-Based Analysis of Transient Interactions between Ketosynthase-like Decarboxylase and Acyl Carrier Protein in a Loading Module of Modular Polyketide Synthase.

Ketosynthase-like decarboxylase (KSQ) domains are widely distributed in the loading modules of modular type I polyketide synthases (PKSs) and catalyze the decarboxylation of the (alkyl-)malonyl unit bound to the acyl carrier protein (ACP) in the loading module for the construction of the PKS starter unit. Previously, we performed a structural and functional analysis of the GfsA KSQ domain involved in the biosynthesis of macrolide antibiotic FD-891. We furthermore revealed the recognition mechanism for the malonic acid thioester moiety of the malonyl-GfsA loading module ACP (ACPL) as a substrate. However, the exact recognition mechanism for the GfsA ACPL moiety remains unclear. Here, we present a structural basis for the interactions between the GfsA KSQ domain and GfsA ACPL. We determined the crystal structure of the GfsA KSQ-acyltransferase (AT) didomain in complex with ACPL (ACPL=KSQAT complex) by using a pantetheine crosslinking probe. We identified the key amino acid residues involved in the KSQ domain-ACPL interactions and confirmed the importance of these residues by mutational analysis. The binding mode of ACPL to the GfsA KSQ domain is similar to that of ACP to the ketosynthase domain in modular type I PKSs. Furthermore, comparing the ACPL=KSQAT complex structure with other full-length PKS module structures provides important insights into the overall architectures and conformational dynamics of the type I PKS modules.

Unique Initiation and Termination Mechanisms Involved in the Biosynthesis of a Hybrid Polyketide-Nonribosomal Peptide Lyngbyapeptin B Produced by the Marine Cyanobacterium Moorena bouillonii.

Lyngbyapeptin B is a hybrid polyketide-nonribosomal peptide isolated from particular marine cyanobacteria. In this report, we carried out genome sequence analysis of a producer cyanobacterium Moorena bouillonii to understand the biosynthetic mechanisms that generate the unique structural features of lyngbyapeptin B, including the (E)-3-methoxy-2-butenoyl starter unit and the C-terminal thiazole moiety. We identified a putative lyngbyapeptin B biosynthetic (lynB) gene cluster comprising nine open reading frames that include two polyketide synthases (PKSs: LynB1 and LynB2), four nonribosomal peptide synthetases (NRPSs: LynB3, LynB4, LynB5, and LynB6), a putative nonheme diiron oxygenase (LynB7), a type II thioesterase (LynB8), and a hypothetical protein (LynB9). In vitro enzymatic analysis of LynB2 with methyltransferase (MT) and acyl carrier protein (ACP) domains revealed that the LynB2 MT domain (LynB2-MT) catalyzes O-methylation of the acetoacetyl-LynB2 ACP domain (LynB2-ACP) to yield (E)-3-methoxy-2-butenoyl-LynB2-ACP. In addition, in vitro enzymatic analysis of LynB7 revealed that LynB7 catalyzes the oxidative decarboxylation of (4R)-2-methyl-2-thiazoline-4-carboxylic acid to yield 2-methylthiazole in the presence of Fe2+ and molecular oxygen. This result indicates that LynB7 is responsible for the last post-NRPS modification to give the C-terminal thiazole moiety in lyngbyapeptin B biosynthesis. Overall, we identified and characterized a new marine cyanobacterial hybrid PKS-NRPS biosynthetic gene cluster for lyngbyapeptin B production, revealing two unique enzymatic logics.

Structural Insight into the Reaction Mechanism of Ketosynthase-Like Decarboxylase in a Loading Module of Modular Polyketide Synthases.

Ketosynthase-like decarboxylase (KSQ) domains are widely distributed in the loading modules of modular polyketide synthases (PKSs) and are proposed to catalyze the decarboxylation of a malonyl or methylmalonyl unit for the construction of the PKS starter unit. KSQ domains have high sequence similarity to ketosynthase (KS) domains, which catalyze transacylation and decarboxylative condensation in polyketide and fatty acid biosynthesis, except that the catalytic Cys residue of KS domains is replaced by Gln in KSQ domains. Here, we present biochemical analyses of GfsA KSQ and CmiP4 KSQ, which are involved in the biosynthesis of FD-891 and cremimycin, respectively. In vitro analysis showed that these KSQ domains catalyze the decarboxylation of malonyl and methylmalonyl units. Furthermore, we determined the crystal structure of GfsA KSQ in complex with a malonyl thioester substrate analogue, which enabled identification of key amino acid residues involved in the decarboxylation reaction. The importance of these residues was confirmed by mutational analysis. On the basis of these findings, we propose a mechanism of the decarboxylation reaction catalyzed by GfsA KSQ. GfsA KSQ initiates decarboxylation by fixing the substrate in a suitable conformation for decarboxylation. The formation of enolate upon decarboxylation is assisted by two conserved threonine residues. Comparison of the structure of GfsA KSQ with those of KS domains suggests that the Gln residue in the active site of the KSQ domain mimics the acylated Cys residue in the active site of KS domains.

Structural Characterization of Complex of Adenylation Domain and Carrier Protein by Using Pantetheine Cross-Linking Probe.

Adenylation domains (A-domains) are responsible for selective incorporation of carboxylic acid substrates in the biosynthesis of various natural products. Each A-domain must recognize a cognate carrier protein (CP) for functional substrate transfer. The transient interactions between an A-domain and CP have been investigated by using acyl vinylsulfonamide adenosine inhibitors as probes to determine the structures of several A-domain-CP complexes. However, this strategy requires a specific vinylsulfonamide inhibitor that contains an acyl group corresponding to the substrate specificity of a target A-domain in every case. Here, we report an alternative strategy for structural characterization of A-domain-CP complexes. We used a bromoacetamide pantetheine cross-linking probe in combination with a Cys mutation to trap the standalone A-domain-CP complex involved in macrolactam polyketide biosynthesis through a covalent linkage, allowing the determination of the complex structure. This strategy facilitates the structural determination of A-domain-CP complexes.