Syue-Yi Lyu

Interception of teicoplanin oxidation intermediates yields new antimicrobial scaffolds

In the search for new efficacious antibiotics, biosynthetic engineering offers attractive opportunities to introduce minor alterations to antibiotic structures that may overcome resistance. Dbv29, a flavin-containing oxidase, catalyzes the four-electron oxidation of a vancomycin-like glycopeptide to yield A40926. Structural and biochemical examination of Dbv29 now provides insights into residues that govern flavinylation and activity, protein conformation and reaction mechanism. In particular, the serendipitous discovery of a reaction intermediate in the crystal structure led us to identify an unexpected opportunity to intercept the normal enzyme mechanism at two different points to create new teicoplanin analogs. Using this method, we synthesized families of antibiotic analogs with amidated and aminated lipid chains, some of which showed marked potency and efficacy against multidrug resistant pathogens. This method offers a new strategy for the development of chemical diversity to combat antibacterial resistance.

In vitro Characterization of Enzymes Involved in the Synthesis of Nonproteinogenic Residue (2S,3S)‐β‐Methylphenylalanine in Glycopeptide Antibiotic Mannopeptimycin

Mannopeptimycin, a potent drug lead, has superior activity against difficult‐to‐treat multidrug‐resistant Gram‐positive pathogens such as methicillin‐resistant Staphylococcus aureus (MRSA). (2S,3S)‐β‐Methylphenylalanine is a residue in the cyclic hexapeptide core of mannopeptimycin, but the synthesis of this residue is far from clear. We report here on the reaction order and the stereochemical course of reaction in the formation of (2S,3S)‐β‐methylphenylalanine. The reaction is executed by the enzymes MppJ and TyrB, an S‐adenosyl methionine (SAM)‐dependent methyltransferase and an (S)‐aromatic‐amino‐acid aminotransferase, respectively. Phenylpyruvic acid is methylated by MppJ at its benzylic position at the expense of one equivalent of SAM. The resulting β‐methyl phenylpyruvic acid is then converted to (2S,3S)‐β‐methylphenylalanine by TyrB. MppJ was further determined to be regioselective and stereoselective in its catalysis of the formation of (3S)‐β‐methylphenylpyruvic acid. The binding constant (KD) of MppJ versus SAM is 26 μM. The kinetic constants with respect to kcat Ppy and KM Ppy, and kcat SAM and KM SAM are 0.8 s−1 and 2.5 mM, and 8.15 s−1 and 0.014 mM, respectively. These results suggest SAM has higher binding affinity for MppJ than Ppy, and the CC bond formation in βmPpy might be the rate‐limiting step, as opposed to the CS bond breakage in SAM.

Regioselective deacetylation based on teicoplanin-complexed Orf2* crystal structures

Lipoglycopeptide antibiotics are more effective than vancomycin against MRSA as they carry an extra aliphatic acyl side chain on glucosamine (Glm) at residue 4 (r4). The biosynthesis of the r4 N-acyl Glc moiety at teicoplanin (Tei) or A40926 has been elucidated, in which the primary amine nucleophile of Glm is freed from the r4 GlcNac pseudo-Tei precursor by Orf2* for the subsequent acylation reaction to occur. In this report, two Orf2* structures in complex with β-D-octyl glucoside or Tei were solved. Of the complexed structures, the substrate binding site and a previously unknown hydrophobic cavity were revealed, wherein r4 GlcNac acts as the key signature for molecular recognition and the cavity allows substrates carrying longer acyl side chains in addition to the acetyl group. On the basis of the complexed structures, a triple-mutation mutant S98A/V121A/F193Y is able to regioselectively deacetylate r6 GlcNac pseudo-Tei instead of that at r4. Thereby, novel analogs can be made at the r6 sugar moiety.

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 β-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.

Combining biocatalysis and chemoselective chemistries for glycopeptide antibiotics modification

Glycopeptide antibiotics are clinically important medicines to treat serious Gram-positive bacterial infections. The emergence of glycopeptide resistance among pathogens has motivated considerable interest in expanding structural diversity of glycopeptide to counteract resistance. The complex structure of glycopeptide poses substantial barriers to conventional chemical methods for structural modifications. By contrast, biochemical approaches have attracted great attention because ample biosynthetic information and sophisticated toolboxes have been made available to change reaction specificity through protein engineering, domain swapping, pathway engineering, addition of substrate analogs, and mutagenesis.

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)-β-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 βMePpy. 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 NATs 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.

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.

Chain Elongation and Cyclization in Type III PKS DpgA

Chain elongation and cyclization of precursors of dihydroxyphenylacetyl‐CoA (DPA‐CoA) catalyzed by the bacterial type III polyketide synthase DpgA were studied. Two labile intermediates, di‐ and tri‐ketidyl‐CoA (DK‐ and TK‐CoA), were proposed and chemically synthesized. In the presence of DpgABD, each of these with [13C3]malonyl‐CoA (MA‐CoA) was able to form partially 13C‐enriched DPA‐CoA. By NMR and MS analysis, the distribution of 13C atoms in the partially 13C‐enriched DPA‐CoA shed light on how the polyketide chain elongates and cyclizes in the DpgA‐catalyzed reaction. Polyketone intermediates elongate in a manner different from that which had been believed: two molecules of DK‐CoA, or one DK‐CoA plus one acetoacetyl‐CoA (AA‐CoA), but not two molecules of AA‐CoA can form one molecule of DPA‐CoA. As a result, polyketidyl‐CoA serves as both the starter and extender, whereas polyketone‐CoA without the terminal carboxyl group can only act as an extender. The terminal carboxyl group is crucial for the cyclization that likely takes place on CoA.

Evidence of Diradicals Involved in the Yeast Transketolase Catalyzed Keto-Transferring Reactions

Transketolase (TK) catalyzes a reversible transfer of a two‐carbon (C2) unit between phosphoketose donors and phosphoaldose acceptors, for which the group‐transfer reaction that follows a one‐ or two‐electron mechanism and the force that breaks the C2“−C3” bond of the ketose donors remain unresolved. Herein, we report ultrahigh‐resolution crystal structures of a TK (TKps) from Pichia stipitis in previously undiscovered intermediate states and support a diradical mechanism for a reversible group‐transfer reaction. In conjunction with MS, NMR spectroscopy, EPR and computational analyses, it is concluded that the enzyme‐catalyzed non‐Kekulé diradical cofactor brings about the C2“−C3” bond cleavage/formation for the C2‐unit transfer reaction, for which suppression of activation energy and activation and destabilization of enzymatic intermediates are facilitated.