Identification of the p-coumaric acid biosynthetic gene cluster in Kutzneria albida: insights into the diazotization-dependent deamination pathway.

Recently, we identified the biosynthetic gene cluster of avenalumic acid (ava cluster) and revealed its entire biosynthetic pathway, resulting in the discovery of a diazotization-dependent deamination pathway. Genome database analysis revealed the presence of more than 100 ava cluster-related biosynthetic gene clusters (BGCs) in actinomycetes; however, their functions remained unclear. In this study, we focused on an ava cluster-related BGC in Kutzneria albida (cma cluster), and revealed that it is responsible for p-coumaric acid biosynthesis by heterologous expression of the cma cluster and in vitro enzyme assays using recombinant Cma proteins. The ATP-dependent diazotase CmaA6 catalyzed the diazotization of both 3-aminocoumaric acid and 3-aminoavenalumic acid using nitrous acid in vitro. In addition, the high efficiency of the CmaA6 reaction enabled us to perform a kinetic analysis of AvaA7, which confirmed that AvaA7 catalyzes the denitrification of 3-diazoavenalumic acid in avenalumic acid biosynthesis. This study deepened our understanding of the highly reducing type II polyketide synthase system as well as the diazotization-dependent deamination pathway for the production of avenalumic acid or p-coumaric acid.

In vitro characterization of nonribosomal peptide synthetase-dependent O-(2-hydrazineylideneacetyl)serine synthesis indicates a stepwise oxidation strategy to generate the α-diazo ester moiety of azaserine.

Azaserine, a natural product containing a diazo group, exhibits anticancer activity. In this study, we investigated the biosynthetic pathway to azaserine. The putative azaserine biosynthetic gene (azs) cluster, which contains 21 genes, including those responsible for hydrazinoacetic acid (HAA) synthesis, was discovered using bioinformatics analysis of the Streptomyces fragilis genome. Azaserine was produced by the heterologous expression of the azs cluster in Streptomyces albus. In vitro enzyme assays using recombinant Azs proteins revealed the azaserine biosynthetic pathway as follows. AzsSPTF and carrier protein (CP) AzsQ are used to synthesize the 2-hydrazineylideneacetyl (HDA) moiety attached to AzsQ from HAA. AzsD transfers the HDA moiety to the C-terminal CP domain of AzsN. The heterocyclization (Cy) domain of the nonribosomal peptide synthetase AzsO synthesizes O-(2-hydrazineylideneacetyl)serine (HDA-Ser) attached to its CP domain from l-serine and HDA moiety-attached AzsN. The thioesterase AzsB hydrolyzes it to yield HDA-Ser, which appears to be converted to azaserine by oxidation. Bioinformatics analysis of the Cy domain of AzsO showed that it has a conserved DxxxxD motif; however, two conserved amino acid residues (Thr and Asp) important for heterocyclization are substituted for Asn. Site-directed mutagenesis of two Asp residues in the DxxxxD motif (D193 and D198) and two substituted Asn residues (N414 and N447) indicated that these four residues are important for ester bond synthesis. These results showed that the diazo ester of azasrine is synthesized by the stepwise oxidation of the HAA moiety and provided another strategy to biosynthesize the diazo group.

Identification and Analysis of the Biosynthetic Gene Cluster for the Hydrazide-Containing Aryl Polyene Spinamycin.

Natural products containing nitrogen-nitrogen (N-N) bonds have attracted much attention because of their bioactivities and chemical features. Several recent studies have revealed the nitrous acid-dependent N-N bond-forming machinery. However, the catalytic mechanisms of hydrazide synthesis using nitrous acid remain unknown. Herein, we focused on spinamycin, a hydrazide-containing aryl polyene produced by Streptomyces albospinus JCM3399. In the S. albospinus genome, we discovered a putative spinamycin biosynthetic gene (spi) cluster containing genes that encode a type II polyketide synthase and genes for the secondary metabolism-specific nitrous acid biosynthesis pathway. A gene inactivation experiment showed that this cluster was responsible for spinamycin biosynthesis. A feeding experiment using stable isotope-labeled sodium nitrite and analysis of nitrous acid-synthesizing enzymes in vitro strongly indicated that one of the nitrogen atoms of the hydrazide group was derived from nitrous acid. In vitro substrate specificity analysis of SpiA3, which is responsible for loading a starter substrate onto polyketide synthase, indicated that N-N bond formation occurs after starter substrate loading. In vitro analysis showed that the AMP-dependent ligase SpiA7 catalyzes the diazotization of an amino group on a benzene ring without a hydroxy group, resulting in a highly reactive diazo intermediate, which may be the key step in hydrazide group formation. Therefore, we propose the overall biosynthetic pathway of spinamycin. This study expands our knowledge of N-N bond formation in microbial secondary metabolism.

Bacterial Avenalumic Acid Biosynthesis Includes Substitution of an Aromatic Amino Group for Hydride by Nitrous Acid Dependent Diazotization.

The diazo group is an important functional group that can confer biological activity to natural products owing to its high reactivity. Recent studies have revealed that diazo groups are synthesized from amino groups using nitrous acid in secondary metabolites of actinomycetes. However, genome database analysis indicated that there are still many diazo group-biosynthesizing enzymes for unknown biosynthetic pathways. Here, we discovered an avenalumic acid biosynthesis gene cluster in Streptomyces sp. RI-77 by genome mining of enzymes involved in diazo group formation. Through heterologous expression, the gene cluster was revealed to direct avenalumic acid (AVA) biosynthesis via 3-aminoavenalumic acid (3-AAA). In vitro enzyme assays showed that AvaA6 and AvaA7 catalyzed the diazotization of 3-AAA using nitrous acid and substitution of the diazo group for hydride to synthesize AVA, respectively. This study revealed an unprecedented pathway for amino group removal via diazotization.

The α/ß Hydrolase AzpM Catalyzes Dipeptide Synthesis in Alazopeptin Biosynthesis Using Two Molecules of Carrier Protein-Tethered Amino Acid.

During the biosynthesis of alazopeptin, a tripeptide composed of two molecules of 6-diazo-5-oxo-L-norleucine (DON) and one of alanine, the α/ß hydrolase AzpM synthesizes the DON-DON dipeptide using DON tethered to the carrier protein AzpF (DON-AzpF). However, whether AzpM catalyzes the condensation of DON-AzpF with DON or DON-AzpF remains unclear. Here, to distinguish between these two condensation possibilities, the reaction catalyzed by AzpM was examined in vitro using a DON analogue, azaserine (AZS). We found that AzpM catalyzed the condensation between AZS-AzpF and DON-AzpF, but not between AZS-AzpF and DON. Possible reaction intermediates, DON-DON-AzpF and AZS-AZS-AzpF, were also detected during AzpM-catalyzed dipeptide formation from DON-AzpF and AZS-AzpF, respectively. From these results, we concluded that AzpM catalyzed the condensation of the two molecules of DON-AzpF and subsequent hydrolysis to produce DON-DON. Thus, AzpM is an unprecedented α/ß hydrolase that catalyzes dipeptide synthesis from two molecules of a carrier protein-tethered amino acid.

Structural and Functional Analyses of the Tridomain-Nonribosomal Peptide Synthetase FmoA3 for 4-Methyloxazoline Ring Formation.

Nonribosomal peptide synthetases (NRPSs) are attractive targets for bioengineering to generate useful peptides. FmoA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), and peptidyl carrier protein (PCP) domains. It uses α-methyl-l-serine to synthesize a 4-methyloxazoline ring, probably with another Cy domain in the preceding module FmoA2. Here, we determined the head-to-tail homodimeric structures of FmoA3 by X-ray crystallography (apo-form, with adenylyl-imidodiphosphate and α-methyl-l-seryl-AMP) and cryogenic electron microscopy single particle analysis, and performed site-directed mutagenesis experiments. The data revealed that α-methyl-l-serine can be accommodated in the active site because of the extra space around Ala688. The Cy domains of FmoA2 and FmoA3 catalyze peptide bond formation and heterocyclization, respectively. FmoA3's Cy domain seems to lose its donor PCP binding activity. The collective data support a proposed catalytic cycle of FmoA3.

Complete Biosynthetic Pathway of Alazopeptin, a Tripeptide Consisting of Two Molecules of 6-Diazo-5-oxo-l-norleucine and One Molecule of Alanine.

DON (6-diazo-5-oxo-l-norleucine), a diazo-containing amino acid, has been studied for more than 60 years as a potent antitumor agent, but its biosynthesis has not been elucidated. Here we reveal the complete biosynthetic pathway of alazopeptin, the tripeptide Ala-DON-DON, which has antitumor activity, by gene inactivation and in vitro analysis of recombinant enzymes. We also established heterologous production of N-acetyl-DON in Streptomyces albus. DON is synthesized from lysine by three enzymes and converted to alazopeptin by five enzymes and one carrier protein. Most interestingly, transmembrane protein AzpL was indicated to catalyze diazotization using 5-oxolysine and nitrous acid as substrates. Site-directed mutagenesis of AzpL indicated that the hydroxy group of Tyr-93 is important for the diazotization. These findings expand our knowledge of the enzymology of N-N bond formation.

Localization of oxidized low-density lipoprotein and its relation to plaque morphology in human coronary artery.

OBJECTIVES: Oxidized low-density lipoprotein (oxLDL) plays a key role in the formation of atherosclerotic plaques. However, its localization in human coronary arterial wall is not well understood. The present study was performed to visualize deposition sites and patterns of native oxLDL and their relation to plaque morphology in human coronary artery. METHODS: Evans blue dye (EB) elicits a violet fluorescence by excitation at 345-nm and emission at 420-nm, and a reddish-brown fluorescence by excitation at 470-nm and emission at 515-nm characteristic of oxLDL only. Therefore, native oxLDL in excised human coronary artery were investigated by color fluorescent microscopy (CFM) using EB as a biomarker. RESULTS: (1) By luminal surface scan with CFM, the % incidence of oxLDL in 38 normal segments, 41 white plaques and 32 yellow plaques that were classified by conventional angioscopy, was respectively 26, 44 and 94, indicating significantly (p<0.05) higher incidence in the latter than the former two groups. Distribution pattern was classified as patchy, diffuse and web-like. Web-like pattern was observed only in yellow plaques with necrotic core. (2) By transected surface scan, oxLDL deposited within superficial layer in normal segments and diffusely within both superficial and deep layers in white and yellow plaques. In yellow plaques with necrotic core, oxLDL deposited not only in the marginal zone of the necrotic core but also in the fibrous cap. CONCLUSION: Taken into consideration of the well-known process of coronary plaque growth, the results suggest that oxLDL begins to deposit in human coronary artery wall before plaque formation and increasingly deposits with plaque growth, exhibiting different deposition sites and patterns depending on morphological changes.

Two-dimensional visualization of cholesterol and cholesteryl esters within human coronary plaques by near-infrared fluorescence angioscopy.

BACKGROUND: Cholesterol (C) and cholesteryl esters (CE) within coronary plaques are minimally visualized directly by any of the available imaging modalities in vivo. If they are rendered visible in vivo, the progression of coronary plaques and the effects of respective therapies on these plaques can be objectively evaluated. HYPOTHESIS: The C and CE within human coronary plaques can be visualized by near-infrared fluorescence angioscopy (NIRFA). METHODS: By exciting at 710 ± 25 nm and emitting at 780 nm, near-infrared fluorescence (NIRF) of lipid components was examined by microscopy in vitro. Lipid components in 49 plaques of 32 excised human coronary arteries were examined by NIRFA in vitro. Coronary plaques were examined by NIRFA in 25 patients with coronary artery disease. RESULTS: C, CE, and calcium (Ca) individually did not exhibit NIRF but did in the presence of ß-carotene, which is known to coexist with lipids in the vascular wall. Other substances that are contained in atherosclerotic plaques did not.² The excised human coronary plaques were classified as those with NIRF and those without. The former plaques were classified into homogenous, doughnut-shaped, and spotty types. Histological examinations revealed that these image patterns were determined by the differences in the locations of C, CE, and Ca, and that those deposited within 700 µm in depth from the plaque surface were imaged by NIRFA. Homogenous, doughnut-shaped, or spotty NIRFA images were also observed in patients. CONCLUSIONS: NIRFA is feasible for 2-dimensional imaging of C and CE deposited in human coronary plaques.

Detection of vulnerable coronary plaques by color fluorescent angioscopy.

OBJECTIVES: This study was carried out to detect vulnerable coronary plaques by color fluorescent angioscopy. BACKGROUND: Collagen fibers (CFs) mainly provide mechanical support to coronary plaques. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation, which in turn destroy CFs while accumulating lipids. As such, demonstration of the absence of CFs, deposition of lipids, and the Ox-LDL may suggest plaque instability. METHODS: Fluorescence of the major components of the atherosclerotic plaques was examined by fluorescent microscopy using a 345-nm band-pass filter and 420-nm band-absorption filter (A-imaging). Fluorescence of Ox-LDL was examined using a 470-nm band-pass filter and 515-nm band-absorption filter (B-imaging) and Evans blue dye as an indicator. Fluorescence in 57 excised human coronary plaques was examined by A-imaging color fluorescent angioscopy. Oxidized LDL in 31 excised coronary plaques and in 12 plaques of 7 patients was investigated by B-imaging color fluorescent angioscopy. RESULTS: Collagen I, collagen IV, and calcium exhibited blue, light blue, and white autofluorescence, respectively. In the presence of beta-carotene which coexists with lipids in the vascular wall, collagen I and IV exhibited green, collagen III and V white, cholesterol yellow, cholesteryl esters orange fluorescence. Oxidized LDL exhibited reddish brown fluorescence in the presence of Evans blue dye. Therefore, coronary plaques exhibited blue, green, white-to-light blue, or yellow-to-orange fluorescence based on plaque composition. Histological examination revealed abundant CFs without lipids in blue plaques; CFs and lipids in green plaques; meager CFs and abundant lipids in white-to-light blue plaques; and the absence of CFs and deposition of lipids, calcium, and macrophage foam cells in the thin fibrous cap in yellow-to-orange plaques, indicating that the yellow-to-orange plaques were most vulnerable. Reddish brown fluorescence characteristic of Ox-LDL was observed in excised coronary plaques, as also in patients. CONCLUSIONS: Color fluorescent angioscopy provides objective information related to coronary plaque composition and may help identify unstable plaques.

Imaging of lysophosphatidylcholine in human coronary plaques by color fluorescence angioscopy.

Lysophosphatidylcholine (LPC) is a proinflammatory and proatherogenic substance, and it plays an important role in the initiation, progression, and destabilization of atherosclerotic plaques. If LPC in the vascular wall is visualized in vivo, the mechanisms of atherosclerosis and the effects of medical and interventional therapies on atherosclerosis can be objectively evaluated. Therefore, this study was carried out to visualize LPC in human coronary plaques using a color fluorescence angioscopy (CFA) system. (1) The fluorescence characteristics of LPC were investigated by color fluorescence microscopy (CFM) using Trypan blue dye (TB) as an indicator. For fluorescence imaging, a combination of a band-pass filter (345 nm) and a band-absorption filter of 420 nm (A imaging), or a combination of a band-pass filter (470 nm) and a band-absorption filter of 520 nm (B imaging) was employed. (2) The fluorescence of LPC in the excised human coronary plaques was investigated by CFA and CFM scanning using the same filters as those in CFM. In the presence of TB, LPC exhibited a red fluorescence in both A and B imaging. This red fluorescence color in both A and B imaging was not observed for the other known major substances that constitute the atherosclerotic plaques. This red fluorescence color in both A and B imaging was detected by CFA in both white and yellow plaques that were classified by conventional angioscopy. This fluorescence color was found to be distributed in a web-like or diffuse configuration by CFM scanning. LPC in the human coronary plaques was successfully visualized by CFA using TB as an indicator.