Cross-Linking of the Nonribosomal Peptide Synthetase Adenylation Domain with a Carrier Protein Using a Pantetheine-Type Probe.

Adenylation domains (A-domains) are responsible for the selective incorporation of carboxylic acid substrates in the biosynthesis of nonribosomal peptides and related natural products. The A-domain transfers an acyl substrate onto its cognate carrier protein (CP). The proper interactions between an A-domain and the cognate CP are important for functional substrate transfer. To stabilize the transient interactions sufficiently for structural analysis of A-domain-CP complex, vinylsulfonamide adenosine inhibitors have been traditionally used as molecular probes. Recently, we have developed an alternative strategy using a synthetic pantetheine-type probe that enables site-specific cross-linking between an A-domain and a CP. In this chapter, we describe the laboratory protocols for this cross-linking reaction.

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.

Recent advances in the structural analysis of adenylation domains in natural product biosynthesis.

Adenylation (A) domains catalyze the biosynthetic incorporation of acyl building blocks into nonribosomal peptides and related natural products by selectively transferring acyl substrates onto cognate carrier proteins (CP). The use of noncanonical acyl units, such as nonproteinogenic amino acids and keto acids, by A domains expands the structural diversity of natural products. Furthermore, interrupted A domains, which have embedded auxiliary domains, are able to modify the incorporated acyl units. Structural information on A domains is important for rational protein engineering to generate unnatural compounds. In this review, we summarize recent advances in the structural analysis of A domains. First, we discuss the mechanisms by which A domains recognize noncanonical acyl units. We then focus on the interactions of A domains with CP domains and embedded auxiliary domains.

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.

One-pot enzymatic synthesis of 2-deoxy-scyllo-inosose from d-glucose and polyphosphate.

2-Deoxy-scyllo-inosose (2DOI, [2S,3R,4S,5R]-2,3,4,5-tetrahydroxycyclohexan-1-one) is a biosynthetic intermediate of 2-deoxystreptamine-containing aminoglycoside antibiotics, including butirosin, kanamycin, and neomycin. In producer microorganisms, 2DOI is constructed from d-glucose 6-phosphate (G6P) by 2-deoxy-scyllo-inosose synthase (DOIS) with the oxidized form of nicotinamide adenine dinucleotide (NAD+). 2DOI is also known as a sustainable biomaterial for production of aromatic compounds and a chiral cyclohexane synthon. In this study, a one-pot enzymatic synthesis of 2DOI from d-glucose and polyphosphate was investigated. First, 3 polyphosphate glucokinases (PPGKs) were examined to produce G6P from d-glucose and polyphosphate. A PPGK derived from Corynebacterium glutamicum (cgPPGK) was found to be suitable for G6P production under ordinary enzymatic conditions. Next, 7 DOISs were examined for the one-pot enzymatic reaction. As a result, cgPPGK and BtrC, the latter of which is a DOIS derived from the butirosin producer Bacillus circulans, achieved nearly full conversion of d-glucose to 2DOI in the presence of polyphosphate.

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.

Generation of incednine derivatives by mutasynthesis.

The macrolactam antibiotic incednine, isolated from Streptomyces sp. ML694-90F3, contains a (S)-3-aminobutyric acid moiety in its polyketide aglycon. In this study, we performed mutasynthesis to generate incednine derivatives. We successfully obtained 28-methylincednine by feeding 3-aminopentanoic acid into culture of a strain in which the glutamate 2,3-aminomutase gene idnL4, whose product is responsible for supplying 3-aminobutyric acid, was disrupted. 28-Methylincednine showed similar suppressive activity of the antiapoptotic function of oncoprotein Bcl-xL to that of incednine. Thus, this study highlights the applicability of the mutasynthesis approach in generation of novel ß-amino acid-containing macrolactam polyketide derivatives.

Stereochemistry in the Reaction of the myo-Inositol Phosphate Synthase Ortholog Ari2 during Aristeromycin Biosynthesis.

The myo-inositol-1-phosphate synthase (MIPS) ortholog Ari2, which is encoded in the aristeromycin biosynthetic gene cluster, catalyzes the formation of five-membered cyclitol phosphate using d-fructose 6-phosphate (F6P) as a substrate. To understand the stereochemistry during the Ari2 reaction in vivo, we carried out feeding experiments with (6S)-d-[6-2H1]- and (6R)-d-[6-2H1]glucose in the aristeromycin-producing strain Streptomyces citricolor. We observed retention of the 2H atom of (6S)-d-[6-2H1]glucose and no incorporation of the 2H atom from (6R)-d-[6-2H1]glucose in aristeromycin. This indicates that Ari2 abstracts the pro-R proton at C6 of F6P after oxidation of C5-OH by nicotinamide adenine dinucleotide (NAD+) to generate the enolate intermediate, which then attacks the C2 ketone to form the C-C bond via aldol-type condensation. The reaction of Ari2 with (6S)-d-[6-2H1]- and (6R)-d-[6-2H1]F6P in vitro exhibited identical stereochemistry compared with that observed during the feeding experiments. Furthermore, analysis of the crystal structure of Ari2, including NAD+ as a ligand, revealed the active site of Ari2 to be similar to that of MIPS of Mycobacterium tuberculosis, supporting the similarity of the reaction mechanisms of Ari2 and MIPS.

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.

Functional and structural characterization of IdnL7, an adenylation enzyme involved in incednine biosynthesis.

Adenylation enzymes play an important role in the selective incorporation of the cognate carboxylate substrates in natural product biosynthesis. Here, the biochemical and structural characterization of the adenylation enzyme IdnL7, which is involved in the biosynthesis of the macrolactam polyketide antibiotic incednine, is reported. Biochemical analysis showed that IdnL7 selects and activates several small amino acids. The structure of IdnL7 in complex with an L-alanyl-adenylate intermediate mimic, 5'-O-[N-(L-alanyl)sulfamoyl]adenosine, was determined at 2.1 Šresolution. The structure of IdnL7 explains the broad substrate specificity of IdnL7 towards small L-amino acids.

An Engineered Aryl Acid Adenylation Domain with an Enlarged Substrate Binding Pocket.

Adenylation (A) domains act as the gatekeepers of non-ribosomal peptide synthetases (NRPSs), ensuring the activation and thioesterification of the correct amino acid/aryl acid building blocks. Aryl acid building blocks are most commonly observed in iron-chelating siderophores, but are not limited to them. Very little is known about the reprogramming of aryl acid A-domains. We show that a single asparagine-to-glycine mutation in an aryl acid A-domain leads to an enzyme that tolerates a wide range of non-native aryl acids. The engineered catalyst is capable of activating non-native aryl acids functionalized with nitro, cyano, bromo, and iodo groups, even though no enzymatic activity of wild-type enzyme was observed toward these substrates. Co-crystal structures with non-hydrolysable aryl-AMP analogues revealed the origins of this expansion of substrate promiscuity, highlighting an enlargement of the substrate binding pocket of the enzyme. Our findings may be exploited to produce diversified aryl acid containing natural products and serve as a template for further directed evolution in combinatorial biosynthesis.

Structural basis of the nonribosomal codes for nonproteinogenic amino acid selective adenylation enzymes in the biosynthesis of natural products.

Nonproteinogenic amino acids are the unique building blocks of nonribosomal peptides (NRPs) and hybrid nonribosomal peptide-polyketides (NRP-PKs) and contribute to their diversity of chemical structures and biological activities. In the biosynthesis of NRPs and NRP-PKs, adenylation enzymes select and activate an amino acid substrate as an aminoacyl adenylate, which reacts with the thiol of the holo form of the carrier protein to afford an aminoacyl thioester as the electrophile for the condensation reaction. Therefore, the substrate specificity of adenylation enzymes is a key determinant of the structure of NRPs and NRP-PKs. Here, we focus on nonproteinogenic amino acid selective adenylation enzymes, because understanding their unique selection mechanisms will lead to accurate functional predictions and protein engineering toward the rational biosynthesis of designed molecules containing amino acids. Based on recent progress in the structural analysis of adenylation enzymes, we discuss the nonribosomal codes of nonproteinogenic amino acid selective adenylation enzymes.

Protein-protein interactions in polyketide synthase-nonribosomal peptide synthetase hybrid assembly lines.

Covering: up to early 2018 Polyketides and nonribosomal peptides are two major families of natural product with a broad range of biological activities. Polyketide synthases (PKSs) assemble small acetic acid-type acyl building blocks into polyketides through C-C bonds, and nonribosomal peptide synthetases (NRPSs) assemble amino acids into peptides through amide bonds. PKS-NRPS hybrid assembly lines build structurally complex polyketide-amino acid/peptide hybrid molecules that incorporate both acyl and aminoacyl building blocks into their products. Their combined functionalities expand the biological activities of these molecules by mixing their chemical properties. Protein-protein interactions are necessary within PKS-NRPS hybrid assembly lines to achieve accurate linkage between the PKS and NRPS systems. This review summarizes the current understanding of the roles and importance of the protein-protein interactions in various PKS-NRPS hybrid assembly lines.

Identification of the Fluvirucin B2 (Sch 38518) Biosynthetic Gene Cluster from Actinomadura fulva subsp. indica ATCC 53714: substrate Specificity of the ß-Amino Acid Selective Adenylating Enzyme FlvN.

Fluvirucins are 14-membered macrolactam polyketides that show antifungal and antivirus activities. Fluvirucins have the ß-alanine starter unit at their polyketide skeletons. To understand the construction mechanism of the ß-alanine moiety in fluvirucin biosyntheses, we have identified the biosynthetic cluster of fluvirucin B2 produced from Actinomadura fulva subsp. indica ATCC 53714. The identified gene cluster contains three polyketide synthases, four characteristic ß-amino acid-carrying enzymes, one decarboxylase, and one amidohydrolase. We next investigated the activity of the adenylation enzyme FlvN, which is a key enzyme for the selective incorporation of a ß-amino acid substrate. FlvN showed strong preference for l-aspartate over other amino acids such as ß-alanine. Based on these results, we propose a biosynthetic pathway for fluvirucin B2.

Biosynthesis of natural products containing ß-amino acids.

Covering: up to January, 2014. We focus here on ß-amino acids as components of complex natural products because the presence of ß-amino acids produces structural diversity in natural products and provides characteristic architectures beyond those of ordinary α-L-amino acids, thus generating significant and unique biological functions in nature. In this review, we first survey the known bioactive ß-amino acid-containing natural products including nonribosomal peptides, macrolactam polyketides, and nucleoside-ß-amino acid hybrids. Next, the biosynthetic enzymes that form ß-amino acids from α-amino acids and the de novo synthesis of ß-amino acids are summarized. Then, the mechanisms of ß-amino acid incorporation into natural products are reviewed. Because it is anticipated that the rational swapping of the ß-amino acid moieties with various side chains and stereochemistries by biosynthetic engineering should lead to the creation of novel architectures and bioactive compounds, the accumulation of knowledge regarding ß-amino acid-containing natural product biosynthetic machinery could have a significant impact in this field. In addition, genome mining of characteristic ß-amino acid biosynthetic genes and unique ß-amino acid incorporation machinery could lead to the discovery of new ß-amino acid-containing natural products.

Characterization of polyphosphate glucokinase SCO5059 from Streptomyces coelicolor A3(2).

SCO5059, encoded in Streptomyces coelicolor A3(2), was identified as a polyphosphate glucokinase. The Km values of SCO5059 for glucose and polyphosphate (poly(P)6) were estimated to be 12 and 4 µM, respectively, and the kcat value was 0.3 s(-1) at pH 7.7 at 28 °C. SCO5059 homologs are highly conserved among Streptomyces, and can work as polyphosphate glucokinase as well.

Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis.

Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the ars operon, are responsible for the biosynthesis of C(22)-C(26) fatty acids from malonyl-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C(22)-C(26) fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyl-CoA by the four members of the ars operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer.

Crystal structure of a family 54 alpha-L-arabinofuranosidase reveals a novel carbohydrate-binding module that can bind arabinose.

As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 alpha-l-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a beta-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a beta-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu(221) and Asp(297), respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.

Mutational and structural analysis of cobalt-containing nitrile hydratase on substrate and metal binding.

Mutants of a cobalt-containing nitrile hydratase (NHase, EC 4.2.1.84) from Pseudonocardia thermophila JCM 3095 involved in substrate binding, catalysis and formation of the active center were constructed, and their characteristics and crystal structures were investigated. As expected from the structure of the substrate binding pocket, the wild-type enzyme showed significantly lower K(m) and K(i) values for aromatic substrates and inhibitors, respectively, than aliphatic ones. In the crystal structure of a complex with an inhibitor (n-butyric acid) the hydroxyl group of betaTyr68 formed hydrogen bonds with both n-butyric acid and alphaSer112, which is located in the active center. The betaY68F mutant showed an elevated K(m) value and a significantly decreased k(cat) value. The apoenzyme, which contains no detectable cobalt atom, was prepared from Escherichia coli cells grown in medium without cobalt ions. It showed no detectable activity. A disulfide bond between alphaCys108 and alphaCys113 was formed in the apoenzyme structure. In the highly conserved sequence motif in the cysteine cluster region, two positions are exclusively conserved in cobalt-containing or iron-containing nitrile hydratases. Two mutants (alphaT109S and alphaY114T) were constructed, each residue being replaced with an iron-containing one. The alphaT109S mutant showed similar characteristics to the wild-type enzyme. However, the alphaY114T mutant showed a very low cobalt content and catalytic activity compared with the wild-type enzyme, and oxidative modifications of alphaCys111 and alphaCys113 residues were not observed. The alphaTyr114 residue may be involved in the interaction with the nitrile hydratase activator protein of P. thermophila.