Structural and functional analysis of l-methionine oxidase identified through sequence data mining.

l-Amino acid oxidase (LAAO), an FAD-dependent enzyme, catalyzes the oxidation of l-amino acids (l-AAs) to their corresponding imino acids. While LAAOs, which can oxidize charged or aromatic l-AAs specifically, have been extensively characterized across various species, LAAOs that have high specificity toward alkyl-chain l-AAs, such as l-Met, are hardly characterized for now. In this study, we screened a highly specific l-Met oxidizing LAAOs from Burkholderiales bacterium (BbMetOx) and Undibacterium sp. KW1 (UndMetOx) using sequence similarity network (SSN) analysis. These enzymes displayed an order of magnitude higher specific activity towards l-Met compared to other l-AAs. Enzyme activity assays showed that these LAAOs operate optimally at moderate condition because the optimal pH and Tm values were pH 7.0 and 58-60°C. We determined the crystal structures of wild-type BbMetOx (BbMetOx(WT)) and an inactivated mutant, BbMetOx (K304A), at 2.7 Å and 2.2 Å resolution, respectively. The overall structure of BbMetOx is closely similar to other known LAAOs of which structures were determined. Comparative analysis of the BbMetOx structures revealed significant conformational changes in the catalytic domain, particularly a movement of approximately 8 Å in the Cα atom of residue Y180. Further analysis highlighted four residues, i.e., Y180, M182, F300, and M302, as critical for l-Met recognition, with alanine substitution at these positions resulting in loss of activity. This study not only underscores the utility of SSN for discovering novel LAAOs but also advances our understanding of substrate specificity in this enzyme family.

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.

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.

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 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.

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.