Biocatalytic Synthesis of Non-Standard Amino Acids by a Decarboxylative Aldol Reaction

The formation of carbon-carbon bonds lies at the heart of organic chemistry, but relatively few C-C bond forming enzymes have found their way into the biocatalysis toolbox. We report that the enzyme UstD performs a highly selective decarboxylative aldol addition with diverse aldehyde substrates to make non-standard, γ-hydroxy amino acids. We increased the activity of UstD through three rounds of classic directed evolution and an additional round of computationally-guided engineering. The enzyme that emerged, UstD2.0, is very efficient in a whole-cell biocatalysis format and readily crystallizes. The X-ray crystal structure of UstD2.0 at 2.25 Å reveals the active site and empowers future studies. The utility of UstD2.0 was demonstrated via the stereoselective gram-scale syntheses of non-standard amino acids.

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Biocatalytic synthesis of non-standard amino acids by a decarboxylative aldol reaction

10.21203/rs.3.rs-544226/v1 ◽

2021 ◽

Author(s):

Andrew Buller ◽

Jonathan Ellis ◽

Meghan Campbell ◽

Prasanth Kumar ◽

Eric Geunes ◽

...

Keyword(s):

Amino Acids ◽

Enzyme Purification ◽

Structural Information ◽

Catalytic Efficiency ◽

X Ray ◽

Bond Forming ◽

Hydroxy Amino ◽

Carbon Carbon Bond ◽

Aldol Addition ◽

Unique Mechanism

Abstract Enzymes are renowned for their catalytic efficiency and selectivity, but relatively few carbon-carbon bond forming enzymes have found their way into the biocatalysis toolbox. While engineering can overcome the challenges associated with C-C bond formation for some enzyme systems, the broader synthetic potential of biocatalysis is hindered by the lack of high-quality C-C bond forming transformations. Here we show that the enzyme UstD performs a highly selective decarboxylative aldol addition with diverse aldehyde substrates to make non-standard, γ-hydroxy amino acids. We increased the activity of UstD through three rounds of classic directed evolution and an additional round of computationally-guided engineering. The enzyme that emerged, UstD2.0, is efficient in a whole-cell biocatalysis format, which circumvents the need for enzyme purification, thereby facilitating its use in traditional organic settings. This new, highly stereoselective enzyme represents a unique expansion of the biosynthetic toolbox. The products are highly desirable, functionally rich bioactive γ-hydroxy amino acids that we demonstrate can be prepared stereoselectively on gram-scale. The X-ray crystal structure of UstD2.0 at 2.25 Å reveals the active site and the molecular basis for the remarkably promiscuity of this catalyst. Taking inspiration from the versatile reactivity of enamines in organic synthesis, we hypothesize that the enamine intermediate of UstD can be engineered to react with electrophiles other than aldehydes. The advent of structural information enabled by engineering of UstD2.0 provides a foundation for probing the unique mechanism of UstD and will guide efforts to expand the reactivity of this unique enzyme.

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The Rosetta all-atom energy function for macromolecular modeling and design

10.1101/106054 ◽

2017 ◽

Cited By ~ 1

Author(s):

Rebecca F. Alford ◽

Andrew Leaver-Fay ◽

Jeliazko R. Jeliazkov ◽

Matthew J. O'Meara ◽

Frank P. DiMaio ◽

...

Keyword(s):

Crystal Structure ◽

Amino Acids ◽

Energy Function ◽

Crystal Structure Data ◽

Structural Data ◽

X Ray ◽

The Past ◽

Biomolecular Modeling ◽

Macromolecular Modeling ◽

Atom Energy

AbstractOver the past decade, the Rosetta biomolecular modeling suite has informed diverse biological questions and engineering challenges ranging from interpretation of low-resolution structural data to design of nanomaterials, protein therapeutics, and vaccines. Central to Rosetta’s success is the energy function: amodel parameterized from small molecule and X-ray crystal structure data used to approximate the energy associated with each biomolecule conformation. This paper describes the mathematical models and physical concepts that underlie the latest Rosetta energy function, beta_nov15. Applying these concepts,we explain how to use Rosetta energies to identify and analyze the features of biomolecular models.Finally, we discuss the latest advances in the energy function that extend capabilities from soluble proteins to also include membrane proteins, peptides containing non-canonical amino acids, carbohydrates, nucleic acids, and other macromolecules.

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Crystal structure of a pyridoxal 5′-phosphate-dependent aspartate racemase derived from the bivalve mollusc Scapharca broughtonii

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x17015813 ◽

2017 ◽

Vol 73 (12) ◽

pp. 651-656 ◽

Cited By ~ 1

Author(s):

Taichi Mizobuchi ◽

Risako Nonaka ◽

Motoki Yoshimura ◽

Katsumasa Abe ◽

Shouji Takahashi ◽

...

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Active Site ◽

Metal Binding ◽

Bivalve Mollusc ◽

Active Site Residues ◽

X Ray ◽

Aspartate Racemase ◽

Scapharca Broughtonii

Aspartate racemase (AspR) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that is responsible for D-aspartate biosynthesis in vivo. To the best of our knowledge, this is the first study to report an X-ray crystal structure of a PLP-dependent AspR, which was resolved at 1.90 Å resolution. The AspR derived from the bivalve mollusc Scapharca broughtonii (SbAspR) is a type II PLP-dependent enzyme that is similar to serine racemase (SR) in that SbAspR catalyzes both racemization and dehydration. Structural comparison of SbAspR and SR shows a similar arrangement of the active-site residues and nucleotide-binding site, but a different orientation of the metal-binding site. Superposition of the structures of SbAspR and of rat SR bound to the inhibitor malonate reveals that Arg140 recognizes the β-carboxyl group of the substrate aspartate in SbAspR. It is hypothesized that the aromatic proline interaction between the domains, which favours the closed form of SbAspR, influences the arrangement of Arg140 at the active site.

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ChemInform Abstract: ORGANOMETALLIC ELECTRON RESERVOIRS. 7. ONE-STEP MULTIPLE FORMATION OF CARBON-CARBON BONDS IN CPFE+(ARENE) SANDWICHES AND UNUSUAL C6ET6 GEOMETRY IN THE X-RAY CRYSTAL STRUCTURE OF CPFE+(η6-C6ET6) PF6-

Chemischer Informationsdienst ◽

10.1002/chin.198314332 ◽

1983 ◽

Vol 14 (14) ◽

Author(s):

J.-R. HAMON ◽

J.-Y. SAILLARD ◽

A. LE BEUZE ◽

M. J. MCGLINCHEY ◽

D. ASTRUC

Keyword(s):

Crystal Structure ◽

X Ray ◽

Carbon Carbon Bonds ◽

Carbon Bonds ◽

One Step

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X-ray studies of crystalline complexes involving amino acids. II. The crystal structure of L-arginine L-glutamate

Acta Crystallographica Section B ◽

10.1107/s0567740877007018 ◽

1977 ◽

Vol 33 (6) ◽

pp. 1754-1759 ◽

Cited By ~ 41

Author(s):

T. N. Bhat ◽

M. Vijayan

Keyword(s):

Crystal Structure ◽

Amino Acids ◽

X Ray

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Difference in Conformation of Virginiamycin M1 in Chloroform and Bound Form in the 50S Ribosome or Streptogramin Acetyltransferase

Australian Journal of Chemistry ◽

10.1071/ch03326 ◽

2004 ◽

Vol 57 (5) ◽

pp. 415 ◽

Cited By ~ 6

Author(s):

Jason Dang ◽

B. Mikael Bergdahl ◽

Frances Separovic ◽

Robert T. C. Brownlee ◽

Robert P. Metzger

Keyword(s):

Crystal Structure ◽

High Resolution ◽

Active Site ◽

Major Change ◽

X Ray ◽

Binding Process ◽

High Resolution Nmr ◽

Virginiamycin M1

The conformation of virginiamycin M1 (VM1) in chloroform, determined by high-resolution NMR experiments, differs significantly from that of the X-ray crystal structure of VM1 bound to the 50S ribosome and to the active site of a streptogramin acetyltransferase enzyme. This implies that the binding process to these entities causes a major change in VM1 conformation.

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Synthesis and Crystal Structure Determination of Cyclosporin H

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc20001317 ◽

2000 ◽

Vol 65 (8) ◽

pp. 1317-1328 ◽

Cited By ~ 7

Author(s):

Alexandr Jegorov ◽

Ladislav Cvak ◽

Aleš Husek ◽

Petr Šimek ◽

Anna Heydová ◽

...

Keyword(s):

Crystal Structure ◽

Amino Acids ◽

Monoclinic Space Group ◽

X Ray Diffraction ◽

Synthesis And Crystal Structure ◽

X Ray ◽

Acid Catalyzed ◽

Products Of Reaction ◽

Cyclosporin H

Acid-catalyzed degradation of cyclosporin A was studied in various solvents and products of reaction were monitored by HPLC. Identification of amino acids and their chirality were determined after hydrolysis and derivatization by GC-MS. Cyclosporin H was isolated as the principal product and its structure was determined by X-ray diffraction: Cyclosporin H- diethyl ether-water (1 : 0.5 : 1) crystallizes in the monoclinic space group I2 with a = 12.338(2) Å, b = 18.963(2) Å, c = 34.074(3) Å, β = 96.47(2)°, Z = 4, and V = 7 921.4(17) Å3.

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Are gamma amino acids promising tools of crystal engineering? – Multicomponent crystals of baclofen

CrystEngComm ◽

10.1039/c5ce01383f ◽

2015 ◽

Vol 17 (43) ◽

pp. 8264-8272 ◽

Cited By ~ 5

Author(s):

Nikoletta B. Báthori ◽

Ornella E. Y. Kilinkissa

Keyword(s):

Crystal Structure ◽

Thermal Analysis ◽

Amino Acids ◽

Crystal Engineering ◽

Sulfonic Acid ◽

Dicarboxylic Acids ◽

Monocarboxylic Acids ◽

X Ray ◽

Toluene Sulfonic Acid

The crystal structure, thermal analysis and powder X-ray analysis of the multicomponent crystals formed between baclofen and selected monocarboxylic acids, dicarboxylic acids and p-toluene sulfonic acid are presented.

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Influence of the presence of the heme cofactor on the JK-loop structure in indoleamine 2,3-dioxygenase 1

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798320013510 ◽

2020 ◽

Vol 76 (12) ◽

pp. 1211-1221

Author(s):

Manon Mirgaux ◽

Laurence Leherte ◽

Johan Wouters

Keyword(s):

Crystal Structure ◽

Cancer Research ◽

Active Site ◽

Loop Structure ◽

X Ray Diffraction ◽

Dynamic Information ◽

X Ray ◽

Indoleamine 2,3 Dioxygenase ◽

First Time ◽

Heme Cofactor

Indoleamine 2,3-dioxygenase 1 has sparked interest as an immunotherapeutic target in cancer research. Its structure includes a loop, named the JK-loop, that controls the orientation of the substrate or inhibitor within the active site. However, little has been reported about the crystal structure of this loop. In the present work, the conformation of the JK-loop is determined for the first time in the presence of the heme cofactor in the active site through X-ray diffraction experiments (2.44 Å resolution). Molecular-dynamics trajectories were also obtained to provide dynamic information about the loop according to the presence of cofactor. This new structural and dynamic information highlights the importance of the JK-loop in confining the labile heme cofactor to the active site.

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