The Structure-Function Relationship of Human Bleomycin Hydrolase: Mutation of a Cysteine Protease into a Serine Protease.

Human bleomycin hydrolase (hBH) catalyzes deamidation of the anticancer drug bleomycins (BLM). This enzyme is involved in BLM detoxification and drug resistance. Herein, we report the putative BLM-binding site and catalytic mechanism of hBH. The crystal structures and biochemical studies suggest that hBH cleaves its C-terminal residue without significant preference for the type of amino acid, and therefore can accordingly accommodate the ß-aminoalanine amide moiety of BLM for deamidation. Interestingly, hBH is capable of switching from a cysteine protease to a serine protease that is unable to cleave the secondary amide of hBH C-terminus but reacts with the primary amide of BLMs.

Characterization and Structural Determination of CmnG-A, the Adenylation Domain That Activates the Nonproteinogenic Amino Acid Capreomycidine in Capreomycin Biosynthesis.

Capreomycidine (Cap) is a nonproteinogenic amino acid and building block of nonribosomal peptide (NRP) natural products. We report the formation and activation of Cap in capreomycin biosynthesis. CmnC and CmnD catalyzed hydroxylation and cyclization, respectively, of l-Arg to form l-Cap. l-Cap is then adenylated by CmnG-A before being incorporated into the nonribosomal peptide. The co-crystal structures of CmnG-A with l-Cap and adenosine nucleotides provide insights into the specificity and engineering opportunities of this unique adenylation domain.

Engineering a High-Affinity Anti-Methoxy Poly(ethylene glycol) (mPEG) Antibody for Sensitive Immunosensing of mPEGylated Therapeutics and mPEG Molecules.

Sensitive quantification of methoxy poly(ethylene glycol) (mPEG)-conjugated therapeutics for pharmacokinetic determination is critical for mPEGylated drug development. However, sensitive measurement of low-molecular-weight (lmw) mPEG compounds remains challenging due to epitope competition between backbone-specific anti-PEG antibodies. Here, we engineered a high-affinity methoxy-specific anti-mPEG antibody for sensitive quantification of free mPEG molecules and mPEGylated therapeutics. The affinity-enhanced h15-2Y antibody variant shows a 10.3-fold increase in mPEG-binding activity compared to parental h15-2b. h15-2Y-based sandwich ELISA can effectively quantify lmw mPEG5K and high-molecular-weight (hmw) mPEG20K at concentrations as low as 3.4 and 5.1 ng mL-1, respectively. Moreover, lmw mPEG compounds (560, 750, 1000, and 2000 Da) can be efficiently quantified via h15-2Y-based competitive ELISA with detection limits at nanomolar levels. This study provides a promising approach for application in the quantitative analysis of the various sizes of mPEG molecules to accelerate the timeline of mPEG-conjugated drug development.

Biomimetic Total Synthesis of the Spiroindimicin Family of Natural Products.

A unified strategy for the biomimetic total synthesis of the spiroindimicin family of natural products was reported. Key transformations include a one-pot two-enzyme-catalyzed oxidative dimerization of L-tryptophan/5-chloro-L-tryptophan to afford the bis-indole precursors chromopyrrolic acid/5',5''-dichloro-chromopyrrolic acid, and regioselective C3'-C2'' and C3'-C4'' bond formation converting a common bis-indole skeleton to two skeletally different natural products, including (±)-spiroindimicins D and G with a [5,5] spiro-ring skeleton, and (±)-spiroindimicins A and H with a [5,6] spiro-ring skeleton, respectively.

Characterization of Enzymes Catalyzing the Formation of the Nonproteinogenic Amino Acid l-Dap in Capreomycin Biosynthesis.

Capreomycin (CMN) and viomycin (VIO) are nonribosomal peptide antituberculosis antibiotics, the structures of which contain four nonproteinogenic amino acids, including l-2,3-diaminopropionic acid (l-Dap), ß-ureidodehydroalanine, l-capreomycidine, and ß-lysine. Previous bioinformatics analysis suggested that CmnB/VioB and CmnK/VioK participate in the formation of l-Dap; however, the real substrates of these enzymes are yet to be confirmed. We herein show that starting from O-phospho-l-Ser (OPS) and l-Glu precursors, CmnB catalyzes the condensation reaction to generate a metabolite intermediate N-(1-amino-1-carboxyl-2-ethyl)glutamic acid (ACEGA), which undergoes NAD+-dependent oxidative hydrolysis by CmnK to generate l-Dap. Furthermore, the binding site of ACEGA and the catalytic mechanism of CmnK were elucidated with the assistance of three crystal structures, including those of apo-CmnK, the NAD+-CmnK complex, and CmnK in an alternative conformation. The CmnK-ACEGA docking model revealed that the glutamate α-hydrogen points toward the nicotinamide moiety. It provides evidence that the reaction is dependent on hydride transfer to form an imine intermediate, which is subsequently hydrolyzed by a water molecule to produce l-Dap. These findings modify the original proposed pathway and provide insights into l-Dap formation in the biosynthesis of other related natural products.

Terpene synthases in disguise: enzymology, structure, and opportunities of non-canonical terpene synthases.

Covering: up to July 2019 Terpene synthases (TSs) are responsible for generating much of the structural diversity found in the superfamily of terpenoid natural products. These elegant enzymes mediate complex carbocation-based cyclization and rearrangement cascades with a variety of electron-rich linear and cyclic substrates. For decades, two main classes of TSs, divided by how they generate the reaction-triggering initial carbocation, have dominated the field of terpene enzymology. Recently, several novel and unconventional TSs that perform TS-like reactions but do not resemble canonical TSs in sequence or structure have been discovered. In this review, we identify 12 families of non-canonical TSs and examine their sequences, structures, functions, and proposed mechanisms. Nature provides a wide diversity of enzymes, including prenyltransferases, methyltransferases, P450s, and NAD+-dependent dehydrogenases, as well as completely new enzymes, that utilize distinctive reaction mechanisms for TS chemistry. These unique non-canonical TSs provide immense opportunities to understand how nature evolved different tools for terpene biosynthesis by structural and mechanistic characterization while affording new probes for the discovery of novel terpenoid natural products and gene clusters via genome mining. With every new discovery, the dualistic paradigm of TSs is contradicted and the field of terpene chemistry and enzymology continues to expand.

Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme.

Acyl-coenzyme A (CoA) ligases catalyze the activation of carboxylic acids via a two-step reaction of adenylation followed by thioesterification. Here, we report the discovery of a non-adenylating acyl-CoA ligase PtmA2 and the functional separation of an acyl-CoA ligase reaction. Both PtmA1 and PtmA2, two acyl-CoA ligases from the biosynthetic pathway of platensimycin and platencin, are necessary for the two steps of CoA activation. Gene inactivation of ptmA1 and ptmA2 resulted in the accumulation of free acid and adenylate intermediates, respectively. Enzymatic and structural characterization of PtmA2 confirmed its ability to only catalyze thioesterification. Structural characterization of PtmA2 revealed it binds both free acid and adenylate substrates and undergoes the established mechanism of domain alternation. Finally, site-directed mutagenesis restored both the adenylation and complete CoA activation reactions. This study challenges the currently accepted paradigm of adenylating enzymes and inspires future investigations on functionally separated acyl-CoA ligases and their ramifications in biology.

Structural Insights into the Free-Standing Condensation Enzyme SgcC5 Catalyzing Ester-Bond Formation in the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027.

C-1027 is a chromoprotein enediyne antitumor antibiotic, consisting of the CagA apoprotein and the C-1027 chromophore. The C-1027 chromophore features a nine-membered enediyne core appended with three peripheral moieties, including an ( S)-3-chloro-5-hydroxy-ß-tyrosine. In a convergent biosynthesis of the C-1027 chromophore, the ( S)-3-chloro-5-hydroxy-ß-tyrosine moiety is appended to the enediyne core by the free-standing condensation enzyme SgcC5. Unlike canonical condensation domains from the modular nonribosomal peptide synthetases that catalyze amide-bond formation, SgcC5 catalyzes ester-bond formation, as demonstrated in vitro, between SgcC2-tethered ( S)-3-chloro-5-hydroxy-ß-tyrosine and ( R)-1-phenyl-1,2-ethanediol, a mimic of the enediyne core as an acceptor substrate. Here, we report that (i) genes encoding SgcC5 homologues are widespread among both experimentally confirmed and bioinformatically predicted enediyne biosynthetic gene clusters, forming a new clade of condensation enzymes, (ii) SgcC5 shares a similar overall structure with the canonical condensation domains but forms a homodimer in solution, the active site of which is located in a cavity rather than a tunnel typically seen in condensation domains, and (iii) the catalytic histidine of SgcC5 activates the 2-hydroxyl group, while a hydrogen-bond network in SgcC5 prefers the R-enantiomer of the acceptor substrate, accounting for the regio- and stereospecific ester-bond formation between SgcC2-tethered ( S)-3-chloro-5-hydroxy-ß-tyrosine and ( R)-1-phenyl-1,2-ethanediol upon acid-base catalysis. These findings expand the catalytic repertoire and reveal new insights into the structure and mechanism of condensation enzymes.

PokMT1 from the Polyketomycin Biosynthetic Machinery of Streptomyces diastatochromogenes Tü6028 Belongs to the Emerging Family of C-Methyltransferases That Act on CoA-Activated Aromatic Substrates.

Recent biochemical characterizations of the MdpB2 CoA ligase and MdpB1 C-methyltransferase (C-MT) from the maduropeptin (MDP, 2) biosynthetic machinery revealed unusual pathway logic involving C-methylation occurring on a CoA-activated aromatic substrate. Here we confirmed this pathway logic for the biosynthesis of polyketomycin (POK, 3). Biochemical characterization unambiguously established that PokM3 and PokMT1 catalyze the sequential conversion of 6-methylsalicylic acid (6-MSA, 4) to form 3,6-dimethylsalicylyl-CoA (3,6-DMSA-CoA, 6), which serves as the direct precursor for the 3,6-dimethylsalicylic acid (3,6-DMSA) moiety in the biosynthesis of 3. PokMT1 catalyzes the C-methylation of 6-methylsalicylyl-CoA (6-MSA-CoA, 5) with a kcat of 1.9 min-1 and a Km of 2.2 ± 0.1 µM, representing the most proficient C-MT characterized to date. Bioinformatics analysis of MTs from natural product biosynthetic machineries demonstrated that PokMT1 and MdpB1 belong to a phylogenetic clade of C-MTs that preferably act on aromatic acids. Significantly, this clade includes the structurally characterized enzyme SibL, which catalyzes C-methylation of 3-hydroxykynurenine in its free acid form, using two conserved tyrosine residues for catalysis. A homology model and site-directed mutagenesis suggested that PokMT1 also employs this unusual arrangement of tyrosine residues to coordinate C-methylation but revealed a large cavity capable of accommodating the CoA moiety tethered to 5. CoA activation of the aromatic acid substrate may represent a general strategy that could be exploited to improve catalytic efficiency. This study sets the stage to further investigate and exploit the catalytic utility of this emerging family of C-MTs in biocatalysis and synthetic biology.

Discovery of Alternative Producers of the Enediyne Antitumor Antibiotic C-1027 with High Titers.

The potent cytotoxicity and unique mode of action make the enediyne antitumor antibiotic C-1027 an exquisite drug candidate for anticancer chemotherapy. However, clinical development of C-1027 has been hampered by its low titer from the original producer Streptomyces globisporus C-1027. Here we report three new C-1027 alternative producers, Streptomyces sp. CB00657, CB02329, and CB03608, from The Scripps Research Institute actinomycetes strain collection. Together with the previously disclosed Streptomyces sp. CB02366 strain, four C-1027 alternative producers with C-1027 titers of up to 11-fold higher than the original producer have been discovered. The five C-1027 producers, isolated from distant geographic locations, are distinct Streptomyces strains based on morphology and taxonomy. Pulsed-field gel electrophoresis and Southern analysis of the five C-1027 producers reveal that their C-1027 biosynthetic gene clusters (BGCs) are all located on giant plasmids of varying sizes. The high nucleotide sequence similarity among the five C-1027 BGCs implies that they most likely have evolved from a common ancestor.

The Mesomeric Effect of Thiazolium on non-Kekulé Diradicals in Pichia stipitis Transketolase.

It is theoretically plausible that thiazolium mesomerizes to congeners other than carbene in a low effective dielectric binding site; especially given the energetics and uneven electronegativity of carbene groups. However, such a phenomenon has never been reported. Nine crystal structures of transketolase obtained from Pichia stipitis (TKps) are reported with subatomic resolution, where thiazolium displays an extraordinary ring-bending effect. The bent thiazolium congeners correlate with non-Kekulé diradicals because there is no gain or loss of electrons. In conjunction with biophysical and biochemical analyses, it is concluded that ring bending is a result of tautomerization of thiazolium with its non- Kekulé diradicals, exclusively in the binding site of TKps. The chemophysical properties of these thiazolium mesomers may account for the great variety of reactivities carried out by thiamine-diphosphate-containing (ThDP) enzymes. The stability of ThDP in living systems can be regulated by the levels of substrates, and hydration and dehydration, as well as diradical-mediated oxidative degradation.

Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function.

Covering: up to January 2017Cytochrome P450 enzymes (P450s) are some of the most exquisite and versatile biocatalysts found in nature. In addition to their well-known roles in steroid biosynthesis and drug metabolism in humans, P450s are key players in natural product biosynthetic pathways. Natural products, the most chemically and structurally diverse small molecules known, require an extensive collection of P450s to accept and functionalize their unique scaffolds. In this review, we survey the current catalytic landscape of P450s within the Streptomyces genus, one of the most prolific producers of natural products, and comprehensively summarize the functionally characterized P450s from Streptomyces. A sequence similarity network of >8500 P450s revealed insights into the sequence-function relationships of these oxygen-dependent metalloenzymes. Although only ∼2.4% and <0.4% of streptomycete P450s have been functionally and structurally characterized, respectively, the study of streptomycete P450s involved in the biosynthesis of natural products has revealed their diverse roles in nature, expanded their catalytic repertoire, created structural and mechanistic paradigms, and exposed their potential for biomedical and biotechnological applications. Continued study of these remarkable enzymes will undoubtedly expose their true complement of chemical and biological capabilities.

Crystal Structures of SgcE6 and SgcC, the Two-Component Monooxygenase That Catalyzes Hydroxylation of a Carrier Protein-Tethered Substrate during the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027 in Streptomyces globisporus.

C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-ß-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-ß-tyrosine. This two-component monooxygenase is remarkable for two reasons. (i) SgcE6 specifically reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl carrier protein (PCP)-tethered substrate. To address the molecular details of substrate specificity, we determined the crystal structures of SgcE6 and SgcC at 1.66 and 2.63 Å resolution, respectively. SgcE6 shares a similar ß-barrel fold with the class I HpaC-like flavin reductases. A flexible loop near the active site of SgcE6 plays a role in FAD binding, likely by providing sufficient space to accommodate the AMP moiety of FAD, when compared to that of FMN-utilizing homologues. SgcC shows structural similarity to a few other known FADH2-dependent monooxygenases and sheds light on some biochemically but not structurally characterized homologues. The crystal structures reported here provide insights into substrate specificity, and comparison with homologues provides a catalytic mechanism of the two-component, FADH2-dependent monooxygenase (SgcE6 and SgcC) that catalyzes the hydroxylation of a PCP-tethered substrate.

Structure of the ent-Copalyl Diphosphate Synthase PtmT2 from Streptomyces platensis CB00739, a Bacterial Type II Diterpene Synthase.

Terpenoids are the largest and most structurally diverse family of natural products found in nature, yet their presence in bacteria is underappreciated. The carbon skeletons of terpenoids are generated through carbocation-dependent cyclization cascades catalyzed by terpene synthases (TSs). Type I and type II TSs initiate cyclization via diphosphate ionization and protonation, respectively, and protein structures of both types are known. Most plant diterpene synthases (DTSs) possess three α-helical domains (αßγ), which are thought to have arisen from the fusion of discrete, ancestral bacterial type I TSs (α) and type II TSs (ßγ). Type II DTSs of bacterial origin, of which there are no structurally characterized members, are a missing piece in the structural evolution of TSs. Here, we report the first crystal structure of a type II DTS from bacteria. PtmT2 from Streptomyces platensis CB00739 was verified as an ent-copalyl diphosphate synthase involved in the biosynthesis of platensimycin and platencin. The crystal structure of PtmT2 was solved at a resolution of 1.80 Å, and docking studies suggest the catalytically active conformation of geranylgeranyl diphosphate (GGPP). Site-directed mutagenesis confirmed residues involved in binding the diphosphate moiety of GGPP and identified DxxxxE as a potential Mg(2+)-binding motif for type II DTSs of bacterial origin. Finally, both the shape and physicochemical properties of the active sites are responsible for determining specific catalytic outcomes of TSs. The structure of PtmT2 fundamentally advances the knowledge of bacterial TSs, their mechanisms, and their role in the evolution of TSs.

Crystal Structure of the Zorbamycin-Binding Protein ZbmA, the Primary Self-Resistance Element in Streptomyces flavoviridis ATCC21892.

The bleomycins (BLMs), tallysomycins (TLMs), phleomycin, and zorbamycin (ZBM) are members of the BLM family of glycopeptide-derived antitumor antibiotics. The BLM-producing Streptomyces verticillus ATCC15003 and the TLM-producing Streptoalloteichus hindustanus E465-94 ATCC31158 both possess at least two self-resistance elements, an N-acetyltransferase and a binding protein. The N-acetyltransferase provides resistance by disrupting the metal-binding domain of the antibiotic that is required for activity, while the binding protein confers resistance by sequestering the metal-bound antibiotic and preventing drug activation via molecular oxygen. We recently established that the ZBM producer, Streptomyces flavoviridis ATCC21892, lacks the N-acetyltransferase resistance gene and that the ZBM-binding protein, ZbmA, is sufficient to confer resistance in the producing strain. To investigate the resistance mechanism attributed to ZbmA, we determined the crystal structures of apo and Cu(II)-ZBM-bound ZbmA at high resolutions of 1.90 and 1.65 Å, respectively. A comparison and contrast with other structurally characterized members of the BLM-binding protein family revealed key differences in the protein-ligand binding environment that fine-tunes the ability of ZbmA to sequester metal-bound ZBM and supports drug sequestration as the primary resistance mechanism in the producing organisms of the BLM family of antitumor antibiotics.

Insights into the binding specificity and catalytic mechanism of N-acetylhexosamine 1-phosphate kinases through multiple reaction complexes.

Utilization of N-acetylhexosamine in bifidobacteria requires the specific lacto-N-biose/galacto-N-biose pathway, a pathway differing from the Leloir pathway while establishing symbiosis between humans and bifidobacteria. The gene lnpB in the pathway encodes a novel hexosamine kinase NahK, which catalyzes the formation of N-acetylhexosamine 1-phosphate (GlcNAc-1P/GalNAc-1P). In this report, seven three-dimensional structures of NahK in complex with GlcNAc, GalNAc, GlcNAc-1P, GlcNAc/AMPPNP and GlcNAc-1P/ADP from both Bifidobacterium longum (JCM1217) and B. infantis (ATCC15697) were solved at resolutions of 1.5-2.2 Å. NahK is a monomer in solution, and its polypeptide folds in a crescent-like architecture subdivided into two domains by a deep cleft. The NahK structures presented here represent the first multiple reaction complexes of the enzyme. This structural information reveals the molecular basis for the recognition of the given substrates and products, GlcNAc/GalNAc, GlcNAc-1P/GalNAc-1P, ATP/ADP and Mg(2+), and provides insights into the catalytic mechanism, enabling NahK and mutants thereof to form a choice of biocatalysts for enzymatic and chemoenzymatic synthesis of carbohydrates.

Structure and mechanism of a nonhaem-iron SAM-dependent C-methyltransferase and its engineering to a hydratase and an O-methyltransferase.

In biological systems, methylation is most commonly performed by methyltransferases (MTs) using the electrophilic methyl source S-adenosyl-L-methionine (SAM) via the S(N)2 mechanism. (2S,3S)-ß-Methylphenylalanine, a nonproteinogenic amino acid, is a building unit of the glycopeptide antibiotic mannopeptimycin. The gene product of mppJ from the mannopeptimycin-biosynthetic gene cluster is the MT that methylates the benzylic C atom of phenylpyruvate (Ppy) to give ßMePpy. Although the benzylic C atom of Ppy is acidic, how its nucleophilicity is further enhanced to become an acceptor for C-methylation has not conclusively been determined. Here, a structural approach is used to address the mechanism of MppJ and to engineer it for new functions. The purified MppJ displays a turquoise colour, implying the presence of a metal ion. The crystal structures reveal MppJ to be the first ferric ion SAM-dependent MT. An additional four structures of binary and ternary complexes illustrate the molecular mechanism for the metal ion-dependent methyltransfer reaction. Overall, MppJ has a nonhaem iron centre that bind, orients and activates the α-ketoacid substrate and has developed a sandwiched bi-water device to avoid the formation of the unwanted reactive oxo-iron(IV) species during the C-methylation reaction. This discovery further prompted the conversion of the MT into a structurally/functionally unrelated new enzyme. Through stepwise mutagenesis and manipulation of coordination chemistry, MppJ was engineered to perform both Lewis acid-assisted hydration and/or O-methyltransfer reactions to give stereospecific new compounds. This process was validated by six crystal structures. The results reported in this study will facilitate the development and design of new biocatalysts for difficult-to-synthesize biochemicals.

Multiple complexes of long aliphatic N-acyltransferases lead to synthesis of 2,6-diacylated/2-acyl-substituted glycopeptide antibiotics, effectively killing vancomycin-resistant enterococcus.

Teicoplanin A2-2 (Tei)/A40926 is the last-line antibiotic to treat multidrug-resistant Gram-positive bacterial infections, e.g., methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE). This class of antibiotics is powered by the N-acyltransferase (NAT) Orf11*/Dbv8 through N-acylation on glucosamine at the central residue of Tei/A40926 pseudoaglycone. The NAT enzyme possesses enormous value in untapped applications; its advanced development is hampered largely due to a lack of structural information. In this report, we present eight high-resolution X-ray crystallographic unary, binary, and ternary complexes in order to decipher the molecular basis for NAT's functionality. The enzyme undergoes a multistage conformational change upon binding of acyl-CoA, thus allowing the uploading of Tei pseudoaglycone to enable the acyl-transfer reaction to take place in the occlusion between the N- and C-halves of the protein. The acyl moiety of acyl-CoA can be bulky or lengthy, allowing a large extent of diversity in new derivatives that can be formed upon its transfer. Vancomycin/synthetic acyl-N-acetyl cysteamine was not expected to be able to serve as a surrogate for an acyl acceptor/donor, respectively. Most strikingly, NAT can catalyze formation of 2-N,6-O-diacylated or C6→C2 acyl-substituted Tei analogues through an unusual 1,4-migration mechanism under stoichiometric/solvational reaction control, wherein selected representatives showed excellent biological activities, effectively counteracting major types (VanABC) of VRE.

Biosynthesis of streptolidine involved two unexpected intermediates produced by a dihydroxylase and a cyclase through unusual mechanisms.

Streptothricin-F (STT-F), one of the early-discovered antibiotics, consists of three components, a ß-lysine homopolymer, an aminosugar D-gulosamine, and an unusual bicyclic streptolidine. The biosynthesis of streptolidine is a long-lasting but unresolved puzzle. Herein, a combination of genetic/biochemical/structural approaches was used to unravel this problem. The STT gene cluster was first sequenced from a Streptomyces variant BCRC 12163, wherein two gene products OrfP and OrfR were characterized in vitro to be a dihydroxylase and a cyclase, respectively. Thirteen high-resolution crystal structures for both enzymes in different reaction intermediate states were snapshotted to help elucidate their catalytic mechanisms. OrfP catalyzes an Fe(II) -dependent double hydroxylation reaction converting L-Arg into (3R,4R)-(OH)2 -L-Arg via (3S)-OH-L-Arg, while OrfR catalyzes an unusual PLP-dependent elimination/addition reaction cyclizing (3R,4R)-(OH)2 -L-Arg to the six-membered (4R)-OH-capreomycidine. The biosynthetic mystery finally comes to light as the latter product was incorporation into STT-F by a feeding experiment.

Crystal structure of CmnB involved in the biosynthesis of the nonproteinogenic amino acid L-2,3-diaminopropionic acid.

L-2,3-Diaminopropionic acid (L-Dap) is a nonproteinogenic amino acid that plays as an important role as a building block in the biosynthesis of several natural products, including capreomycin, viomycin, zwittermicin, staphyloferrin and dapdiamide. A previous study reported that CmnB and CmnK are two enzymes that are involved in the formation of L-Dap in the biosynthesis of capreomycin. CmnB catalyzes the condensation reaction of O-phospho-L-serine and L-glutamic acid to generate N-(1-amino-1-carboxyl-2-ethyl)glutamic acid, which subsequently undergoes oxidative hydrolysis via CmnK to generate the product L-Dap. Here, the crystal structure of CmnB in complex with the reaction intermediate PLP-α-aminoacrylate is reported at 2.2 Šresolution. Notably, CmnB is the second known example of a PLP-dependent enzyme that forms a monomeric structure in crystal packing. The crystal structure of CmnB also provides insights into the catalytic mechanism of the enzyme and supports the biosynthetic pathway of L-Dap reported in previous studies.