16 research outputs found
<i>De Novo</i> Biosynthesis of β‑Valienamine in Engineered <i>Streptomyces hygroscopicus</i> 5008
The C<sub>7</sub>N 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<sub>free</sub> 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><sub>IV′</sub>) 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