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Review
. 2017 Apr 26;117(8):5389-5456.
doi: 10.1021/acs.chemrev.6b00623. Epub 2017 Mar 3.

YcaO-Dependent Posttranslational Amide Activation: Biosynthesis, Structure, and Function

Brandon J Burkhart, Christopher J Schwalen, Greg Mann  1 , James H Naismith  1   2 , Douglas A Mitchell
Affiliations
Review

YcaO-Dependent Posttranslational Amide Activation: Biosynthesis, Structure, and Function

Brandon J Burkhart et al. Chem Rev. .

Abstract

With advances in sequencing technology, uncharacterized proteins and domains of unknown function (DUFs) are rapidly accumulating in sequence databases and offer an opportunity to discover new protein chemistry and reaction mechanisms. The focus of this review, the formerly enigmatic YcaO superfamily (DUF181), has been found to catalyze a unique phosphorylation of a ribosomal peptide backbone amide upon attack by different nucleophiles. Established nucleophiles are the side chains of Cys, Ser, and Thr which gives rise to azoline/azole biosynthesis in ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products. However, much remains unknown about the potential for YcaO proteins to collaborate with other nucleophiles. Recent work suggests potential in forming thioamides, macroamidines, and possibly additional post-translational modifications. This review covers all knowledge through mid-2016 regarding the biosynthetic gene clusters (BGCs), natural products, functions, mechanisms, and applications of YcaO proteins and outlines likely future research directions for this protein superfamily.

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Figures

Figure 1
Figure 1
(A) Generic RiPP pathway. (B) Overview of common steps in maturation of RiPP natural products. (C) Example of a posttranslational modification within a RiPP biosynthetic pathway. Here, a peptidic azoline is generated by the ATP-dependent cyclodehydration of a Cys, Ser, or Thr residue. This is the best characterized function for a YcaO protein.
Figure 2
Figure 2
Overview of YcaO superfamily. Asterisk denotes putative functions.
Figure 3
Figure 3
Representative thiazole-containing compounds that are of non-ribosomal or synthetic origin.
Figure 4
Figure 4
Derivatization of the thiopeptide GE2270A enhances water solubility (in parentheses).
Figure 5
Figure 5
Microcin B17 biosynthesis and structure. (A) BGC for MccB17 (B) Precursor peptide sequence of McbA and structure of MccB17. The two bisheterocyclic sites are known as the A- and B-sites. (C) Biosynthetic scheme for the cyclodehydratase/dehydrogenase.
Figure 6
Figure 6
Streptolysin S biosynthetic components. (A) Representative BGCs for members of the SLS-like cytolysin family. (B) Precursor peptide sequences. The leader peptide cleavage site is unknown but suspected to be directly N-terminal to the NPH region in the core region. The final structure for any cytolysin remains unknown.
Figure 7
Figure 7
The synthetic Me2-Arg-Az5 analog of PZN.
Figure 8
Figure 8
Plantazolicin biosynthesis and structure. (A) PZN BGC from Bacillus velezensis. (B) Structure of PZN and sequence alignment of precursor peptides.
Figure 9
Figure 9
Hakacin biosynthetic components. (A) Representative hakacin BGCs (B) Precursor peptide sequences for BalhA1 and BalhA2 with emphasized heterocyclizible residues. The final structure for hakacin is also unknown.
Figure 10
Figure 10
HCA biosynthetic components. (A) Heterocycloanthracin (HCA) gene clusters from related Bacillus organisms (. (B) Sequence of HcaA from B. sp. Al Hakam.
Figure 11
Figure 11
Azolemycin biosynthesis and structure. (A) Azolemycin BGC. (B) Precursor peptide and structure for azolemycin A.
Figure 12
Figure 12
Goadsporin biosynthesis and structure. (A) The goadsporin BGC. (B) Precursor peptide and structure for goadsporin.
Figure 13
Figure 13
Overview of thiopeptide biosynthesis and macrocycle architecture. (A) Thiopeptide BGC highlighting the enzymes that form the minimal thiopeptide scaffold. (B) Structure of micrococcin P1 with hallmark modifications color-coded: YcaO-catalyzed cyclodehydration (blue), lanthipeptide-like dehydrations (purple) and [4+2] cycloaddition (orange). The four-ring motif important for TipA recognition is also highlighted (red). (C) Biosynthesis of the 6-membered nitrogenous heterocycle and corresponding series designations.
Figure 14
Figure 14
Thiopeptide secondary macrocycles. (A) Quinaldic acid derived ring in siomycin (red). (B) Indolic acid derived ring in nosiheptide (blue). (C) Thioether crosslink formed ring in cyclothiazomycin (purple). Attachment points to the main scaffold are highlighted (yellow).
Figure 15
Figure 15
Formation of quinaldic (A) and indolic (B) acid moieties.
Figure 16
Figure 16
Two mechanisms of thiopeptide C-terminal amidation illustrated from nosiheptide (A) and thiostrepton (B) biosynthesis.
Figure 17
Figure 17
Sequence comparison of the core regions for all known thiopeptides. Thiopeptides with the same precursor peptide vary in ancillary tailoring to give different compounds. Groups of related thiopeptides are separated based on azol(in)e position (gray shading), ring size, and secondary ring. Group representatives are in bold font. Azol(in)e, blue; dehydroamino acids, orange; piperidine/pyridine, green; secondary ring, magenta. aName of the most common congener or parent compound. bCore sequences are from BGCs or inferred from the structure of the mature product. cThe number of atoms in the primary macrocycle. dThe moiety that serves to link the secondary side ring to the primary macrocycle. eKnown BGC.
Figure 18
Figure 18
Thiostrepton biosynthesis and structure. (A) Thiostrepton BGC. (B) Precursor peptide and structure for thiostrepton A.
Figure 19
Figure 19
Nosiheptide and nocathiacin biosynthesis and structure. (A) BGCs for nosiheptide and nocathiacin. (B) Precursor peptide and structure of nosiheptide with characteristic, ester-linked indole macrocycle. (C) Precursor peptide and structure of nocathiacin with characteristic glycone and additional oxidations when compared to nosiheptide.
Figure 20
Figure 20
Thiocillin biosynthesis and structure. (A) BGC of thiocillin. (B) Precursor peptide and chemical structure of thiocillin I.
Figure 21
Figure 21
GE2270A and thiomuracin A biosynthesis and structure. (A) BGC for GE2270 and thiomuracin A showing ancillary tailoring enzymes that C-methylate thiazoles and further decorate various side chains. (B) Precursor peptide and structure of GE2270A. (C) Precursor peptide and structure of thiomuracin A.
Figure 22
Figure 22
Cyclothiazomycin biosynthesis and structure. (A) Cyclothiazomycin BGC (B) Cyclothiazomycin A precursor peptide and structure.
Figure 23
Figure 23
Lactazole biosynthesis and structure. (A) The lactazole BGC. (B) Precursor peptide and structure of lactazole A. Stereochemistry has not been rigorously established but each amino acid is presumed to be in natural L configuration.
Figure 24
Figure 24
Berninamycin and TP-1161 biosynthesis and structure. (A) BGCs for berninamycin/TP-1161 (B) Precursor sequence and structure for berninamycin A. (C) Precursor sequence and structure for TP-1161.
Figure 25
Figure 25
Tyrosine C-prenylation. (A) After O-prenylation by a LynF-homolog, a spontaneous Claisen rearragenement generates an ortho C-prenylated product. (B) Example of C-prenylation in the aestuaramide A.
Figure 26
Figure 26
Cyanobactin genotypes I-IV and associated structures. (A) Genotype I. (B) Genotype II. The piricyclamides are likely genotype II, but this has not been strictly classified. (C) Genotype III. (D) Genotype IV.
Figure 27
Figure 27
Linear cyanobactin biosynthesis. (A) BGC for the aeruginosamides. (B) Structure of aeruginosamide B an C.
Figure 28
Figure 28
Cyclodehydratase-associated NHLP and Nif11 biosynthesis and structure. (A) Representative BGCs putatively encoding azol(in)e-containing RiPP products. (B) C-terminal portion of precursor sequences from the NHLP family, highlighting the proposed leader peptide cleavage site is (after VAGG).
Figure 29
Figure 29
YM-216391 biosynthesis and structure. (A) The BGC for YM-216391 (B) Precursor peptide and structure for YM-216391 (C) Structure of marthiapeptide.
Figure 30
Figure 30
Overview of different types of RiPP cyclodehydratases. Until the bottromycin and trifolitoxin YcaOs are reconstituted in vitro, their status as a “standalone YcaO” remains a bioinformatic prediction.
Figure 31
Figure 31
Diagram of active canonical RiPP cyclodehydratases. (A) Crystal structure of LynD (PDB ID: 4VLT). Colored regions represent the YcaO (blue), E1-like (green), Ocin-ThiF-like (orange), leader peptide (yellow), core (black) regions. Dark and light colors denote different monomers. (B) Schematic model of the dimeric cyclodehydratase shown in panel A. (C) Schematic model of the F-dependent cyclodehydratase.
Figure 32
Figure 32
Proposed mechanisms for azoline biosynthesis by YcaO domains. (A) Direct activation suggests the incorporation of phosphate(s) directly into the heterocyclized intermediate, providing a leaving group (Pi) for elimination. (B) The molecular machine hypothesis proposes an allosteric activation role for ATP in which an active conformation of the cyclodehydratase is generated by ATP hydrolysis. (C) An intein-splicing-like mechanism, by which protonation of the hemiorthoamide intermediate allows for elimination of water to generate the azoline in a manner independent of ATP.
Figure 33
Figure 33
Stable isotope labeling experiments support the direct activation mechanism. 31P-NMR was used to monitor the oxygen isotope status of the phosphate byproduct. Cyclodehydratase reactions in [18O]-H2O (top) showed no [18O] enrichment in the phosphate. When [18O]-BalhA was used as the substrate (bottom), the 18O label was incorporated into the free phosphate.
Figure 34
Figure 34
Acidic hydrolysis of thiazoline in [18O]-H2O site-selectively introduces an [18O] label into the carbonyl of the adjacent amino acid. This is the key aspect underlying the AMPL method.
Figure 35
Figure 35
Oxazoles are used in organic synthesis in Diels-Alder [4 + 2] cycloaddition reactions. This chemistry is a feature of a conjugated di-ene, and not of an aromatic ring. Thiazoles, which exhibit greater aromatic nature than oxazoles, do not readily undergo this chemistry.
Figure 36
Figure 36
A plausible mechanism for the dehydrogenation of methyloxazoline catalyzed by B enzymes. The conserved Lys-Tyr motif is likely involved in deprotonating the Cα of the azoline, which is followed by hydride transfer, in what is essentially an E2 mechanism.
Figure 37
Figure 37
A plausible mechanism for cyanobactin epimerization.
Figure 37
Figure 37
A plausible mechanism for cyanobactin epimerization.
Figure 38
Figure 38
Order of biosynthetic enzymes in patellamide biosynthesis. The exact timing of epimerization is not known.
Figure 39
Figure 39
X-ray crystal structure of the dehydrogenase from cyanotheceamide biosynthesis. (A) Structure of the dimeric dehydrogenase or (B) as a single monomer. (C) The FMN molecule (magenta) is bound at the dimer interface. (D) Ligand interaction diagram of FMN binding. Putative hydrogen bonds are shown in green with distances, light red arcs indicate hydrophobic interactions.
Figure 40
Figure 40
Cartoon illustrating cyclodehydratase processing of cysteine residues. The precursor peptide, composed of the leader (green) and core (peptide), can be cyclodehydrated in an ordered fashion that depends on the cyclodehydratase although the preferred order can non-linear as well.
Figure 41
Figure 41
PatE leader peptide binding by LynD. Ordered residues (sticks) of the leader region (yellow) interact with the RRE (green, domain 1). PatE residues are marked with an apostrophe. Presumed salt bridges are shown with dashed lines.
Figure 42
Figure 42
Nucleotide binding in YcaO domains. (A) A ligand interaction diagram for AMPPNP bound to LynD. (B) Residues involved in adenosine and ribose recognition in LynD-AMPPNP (purple) and Ec-YcaO-AMPCPP (green) complex structures. (C) Residues involved in Mg2+ and phosphate coordination in LynD-AMPPNP (purple) and Ec-YcaO-AMPCPP (green) complex structures.
Figure 43
Figure 43
Ribbon structure of dimeric Ec-YcaO. Colored regions represent the YcaO domain (blue), tetratricopeptide-like domain (gray), ATP (orange spheres), or different monomers (dark vs. light coloring).
Figure 44
Figure 44
Bottromycin biosynthesis and structure (A) Bottromycin BGC from Streptomyces bottropensis. (B) Structure of bottromycin and table of congeners. Modifications attributed to YcaOs in red. (C) Sequence alignment of bottromycin precursor peptides.
Figure 45
Figure 45
Biosynthesis of bottromycin determined through gene deletion in a heterologous host (S. coelicolor) and analysis of detected intermediates.
Figure 46
Figure 46
YcaOs catalyze diverse reactions through a similar backbone-activating mechanism. We hypothesize that YcaOs ubiquitously use ATP to O-phosphorylate peptide backbones, generating a common hemiorthoamide intermediate upon nucleophilic attack. Phosphate elimination yields different functional groups dependent on the identity of the nucleophile.
Figure 47
Figure 47
Thioviridamide biosynthesis and structure. (A) The BGC for thioviridamide from Streptomyces olivoviridis. (B) Sequence of precursor peptide TvaA and structure of thioviridamide and analog JBIR-140. Stereochemistry of some JBIR-140 residues was determined by Marfey’s method and assumed to be consistent in thioviridamide.
Figure 48
Figure 48
Trifolitoxin biosynthesis and partial structure. (A) BGC for TFX B. Precursor sequence and partial structure of TFX.
Figure 49
Figure 49
Cartoon schematic of LynD and the engineered ‘activated’ cyclodehydratase (AcLynD). The LynD dimer undergoes a conformational rearrangement (purple and pink) following binding of PatE (black) generating an ‘active’ enzyme. In the absence of leader peptide, the conformational rearrangement is not possible and heterocyclization of the core peptide is inefficient. Covalent attachment of the leader peptide to the enzyme (cyan) in AcLynD mimics the ‘activated’ native LynD, allowing for efficient wild-type-like processing of the core peptide.
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