<i>De Novo</i> Biosynthesis of β‑Valienamine in Engineered <i>Streptomyces hygroscopicus</i> 5008

The C₇N aminocyclitol β-valienamine is a lead compound for the development of new biologically active β-glycosidase inhibitors as chemical chaperone therapeutic agents for lysosomal storage diseases. Its chemical synthesis is challenging due to the presence of multichiral centers in the structure. Herein, we took advantage of a heterogeneous aminotransferase with stereospecificity and designed a novel pathway for producing β-valienamine in <i>Streptomyces hygroscopicus</i> 5008, a validamycin producer. The aminotransferase BtrR from <i>Bacillus circulans</i> was able to convert valienone to β-valienamine with an optical purity of up to >99.9% enantiomeric excess value <i>in vitro</i>. When the aminotransferase gene was introduced into a mutant of <i>S. hygroscopicus</i> 5008 accumulating valienone, 20 mg/L of β-valienamine was produced after 96 h cultivation in shaking flasks. This work provides a powerful alternative for preparing the chiral intermediates for pharmaceutical development

Stereospecificity of Enoylreductase Domains from Modular Polyketide Synthases

An enoylreductase (ER) domain of a polyketide synthase module recruiting a methylmalonate extender unit sets the C2 methyl branch to either the <i>S</i> or <i>R</i> configuration during processing of a polyketide intermediate carried by an acyl carrier protein (ACP) domain. In the present study, pantetheine- and ACP-bound <i>trans</i>-2-methylcrotonyl substrate surrogates were used to scrutinize the stereospecificity of the ER domains. The pantetheine-bound thioester was reduced to mixtures of both 2<i>R</i> and 2<i>S</i> products, whereas the expected 2<i>S</i> epimer was almost exclusively generated when the cognate ACP-bound substrate surrogate was utilized. The analogous incubation of an ER with the substrate surrogate carried by a noncognate ACP significantly increased the generation of the unexpected 2<i>R</i> epimer, highlighting the dependence of stereospecificity on proper protein–protein interactions between ER and ACP domains. The ER mutant assays revealed the involvement of the conserved tyrosine and lysine in stereocontrol. Taken together, these results expand the current understanding of the ER stereochemistry and help in the engineering of modular PKSs

Superposition of OtsA and ValL structures.

<p>(a) Ribbon diagram overlap. The ribbon diagram of OtsA (colored in blue) is superposed with that of ValL(colored in green). The boxed region shows the main difference between two structures. (b) Active site overlap. The active site residues responsible for V7P binding in OtsA (colored in blue) are superposed with the active site residues in ValL(colored in green). Residue-labels in ValL are shown.</p

The proposed mechanism for ValL.

<p>Adopted from the mechanism of OtsA, a SNi-like mechanism is proposed for ValL. The transition state is enclosed in brackets and GMP is designated as X.</p

Structure-based sequence alignment of ValL and OtsA from several species.

<p>The primary sequence of ValL is aligned with the OtsA sequences from <i>E. coli</i>, <i>Mycobacterium tuberculosis</i>, and <i>Candida albicans</i>. The sequences are annotated with corresponding secondary structures in ValL. Arrows represent β-sheets and helices represent α-helices. The active-site loops are shaded and the conserved residues are colored in red. The residues involved in binding of V7P to OtsA are marked by .</p

Data collection and refinement statistics.

1<p>Data in the parenthesis was calculated based on the highest resolution shell.</p>2<p>Mean figure of merit with or without density modification.</p>3<p>R-factor = (Σhkl||Fo|-|Fc||)/Σhkl|Fo| where Fo and Fc are the observed and calculated structure factors respectively. R_free was calculated with a randomly-selected 5% subset which was excluded from the refinement process.</p>4<p>Statistics of Ramachandran plot were calculated with MolProbity.</p

Reactions catalyzed by OtsA, ValL and chemical structures of related natural products.

<p>(a) The reaction catalyzed by OtsA. (b) The reaction catalyzed by ValL. (c) Chemical structures of validoxyamine A, trehalose and validamycin A.</p

Theoretical Studies on the Mechanism of Thioesterase-Catalyzed Macrocyclization in Erythromycin Biosynthesis

Macrocyclic polyketides, biosynthesized by modular polyketide synthases (PKSs), have been developed successfully into generation-by-generation pharmaceuticals for numerous therapeutic areas. A great effort has been made experimentally and theoretically to elucidate the biosynthesis mechanisms, in particular for thioesterase (TE)-mediated macrocyclization, which controls the final step in the PKS biosynthesis and determines chemical structures of the final products. To obtain a better insight into the macrocyclization process (i.e., releasing step), we carried out MD simulations, QM and QM/MM calculations on complexes of 6-deoxyerythronolide B synthase (DEBS) TE and two substrates, one toward a macrocyclic product and another toward a linearly hydrolytic product. Our investigation showed the induced-fit mutual recognition between the TE enzyme and substrates: in the case of macrocyclization, a critical hydrogen-bonding network is formed between the enzyme and substrate <b>1</b>, and a hydrophobic pocket appropriately accommodates the substrate in the lid region, in which a pivotal prereaction state (<b>1</b>_IV′) with an energy barrier of 11.6 kcal/mol was captured on the potential energy surface calculation. Accompanied with the deprotonation of the prereaction state, the nucleophilic attack occurs with a calculated barrier of 9.9 kcal/mol and leads to the charged tetrahedral intermediate. Following the decomposition of the intermediate, the final macrocyclic product releases with a relatively low barrier. However, in the case of hydrolysis, such a prereaction state for cyclization was not observed in similar molecular simulations. These calculations are consistent with the previous biochemical and structural studies about the TE-mediated reactions. Our study indicated that the enzyme–substrate specificity stems from mutual molecular recognition via a prereaction state between DEBS TE and substrates, suggesting a prereaction-and-action mechanism in the TE macrocyclization and release of PKS product

Modeling of V7P in the ValL active site.

<p>The V7P molecule is modeled in the active site of ValL using the docking program AutoDock. The ValL/V7P complex model (colored in green) is superposed with OtsA/V7P complex structure (colored in blue). The V7P molecules are shown in cylinders and the proteins are shown in ribbons.</p

Three-dimensional structures of ValL and GT-B glycosyltransferases.

<p>A diagram showing N-terminal domain (green), C-terminal domain (blue) and the cleft harboring the active site. (a) ValL. (b) OtsA. (c) T4 β-glucosyltransferase.</p