I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598, 24948]
Open Access
Abstract: Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) is a sulfosugar that is the anionic head group of plant, algal and cyanobacterial sulfolipids: sulfoquinovosyl diacylglycerols. SQ is produced within photosynthetic tissues, forms a major terrestrial reservoir of biosulfur, and is an important species within the biogeochemical sulfur cycle. A major pathway for SQ breakdown is the sulfoglycolytic Embden-Meyerhof-Parnas (sulfo-EMP) pathway, which involves cleavage of the 6-carbon chain of the intermediate sulfofructose-1-phosphate (SFP) into dihydroxyacetone and sulfolactaldehyde, catalyzed by class I or II SFP aldolases. While the molecular basis of catalysis is understood for class I SFP aldolases, comparatively little is known about class II SFP aldolases. Here, we report the molecular architecture and biochemical basis of catalysis of two metal-dependent class II SFP aldolases from Hafnia paralvei and Yersinia aldovae. 3D X-ray structures of complexes with substrate SFP and product DHAP reveal a dimer-of-dimers (tetrameric) assembly, the sulfonate binding pocket, two metal binding sites, and flexible loops that are implicated in catalysis. Both enzymes were metal dependent and exhibited high KM values for SFP, consistent with their role in a unidirectional nutrient acquisition pathway. Bioinformatic analysis identified a range of sulfo-EMP gene clusters containing class I/II SFP aldolases. The class I and II SFP aldolases occur exclusively within Actinobacteria and Firmicutes phyla, respectively, while both classes of enzyme occur within Proteobacteria. This work emphasizes the importance of SQ as a nutrient for diverse bacterial phyla and the different chemical strategies they use to harvest carbon from this sulfosugar.
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Oct 2023
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: The NADPH-dependent imine reductase from Ajellomyces dermatitidis (AdRedAm) catalyzes the reductive amination of certain ketones with amine donors supplied in an equimolar ratio. The structure of AdRedAm has been determined in three forms. The first form, which belongs to space group P3121 and was refined to 2.01 Å resolution, features two molecules (one dimer) in the asymmetric unit in complex with the redox-inactive cofactor NADPH4. The second form, which belongs to space group C21 and was refined to 1.73 Å resolution, has nine molecules (four and a half dimers) in the asymmetric unit, each complexed with NADP+. The third form, which belongs to space group P3121 and was refined to 1.52 Å resolution, has one molecule (one half-dimer) in the asymmetric unit. This structure was again complexed with NADP+ and also with the substrate 2,2-difluoroacetophenone. The different data sets permit the analysis of AdRedAm in different conformational states and also reveal the molecular basis of stereoselectivity in the transformation of fluorinated acetophenone substrates by the enzyme.
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Sep 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Sulfoquinovose (SQ) is a key component of plant sulfolipids (sulfoquinovosyl diacylglycerols) and a major environmental reservoir of biological sulfur. Breakdown of SQ is achieved by bacteria through the pathways of sulfoglycolysis. The sulfoglycolytic sulfofructose transaldolase (sulfo-SFT) pathway is used by gut-resident firmicutes and soil saprophytes. After isomerization of SQ to sulfofructose (SF), the namesake enzyme catalyzes the transaldol reaction of SF transferring dihydroxyacetone to 3C/4C acceptors to give sulfolactaldehyde and fructose-6-phosphate or sedoheptulose-7-phosphate. We report the 3D cryo-EM structure of SF transaldolase from Bacillus megaterium in apo and ligand bound forms, revealing a decameric structure formed from two pentameric rings of the protomer. We demonstrate a covalent “Schiff base” intermediate formed by reaction of SF with Lys89 within a conserved Asp-Lys-Glu catalytic triad and defined by an Arg-Trp-Arg sulfonate recognition triad. The structural characterization of the signature enzyme of the sulfo-SFT pathway provides key insights into molecular recognition of the sulfonate group of sulfosugars.
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Feb 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Sulfoquinovose (SQ) is the anionic headgroup of the ubiquitous plant sulfolipid, sulfoquinovosyl diacylglycerol (SQDG). SQDG can undergo delipidation to give sulfoquinovosyl glycerol (SQGro) and further glycoside cleavage to give SQ, which can be metabolized through microbial sulfoglycolytic pathways. Exogenous SQDG metabolites are imported into bacteria through membrane spanning transporter proteins. The recently discovered sulfoglycolytic sulfoquinovose monooxygenase (sulfo-SMO) pathway in Agrobacterium tumefaciens features a periplasmic sulfoquinovosyl glycerol binding protein, SmoF, and an ATP-binding cassette (ABC) transporter. Here, we use X-ray crystallography, differential scanning fluorimetry and isothermal titration calorimetry to study SQ glycoside recognition by SmoF. This work reveals that in addition to SQGro, SmoF can also bind SQ, a simple methyl glycoside and even a short-chain SQDG analogue. Molecular recognition of these substrates is achieved through conserved interactions with the SQ-headgroup together with more plastic interactions with the aglycones. This suggests that the solute binding protein of A. tumefaciens, and related SQ-binding proteins from other sulfoglycolytic pathways, can provide their host organisms direct access to most of the SQ metabolites known to be produced by phototrophs.
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Mar 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Mahima
Sharma
,
James P.
Lingford
,
Marija
Petricevic
,
Alexander J. D.
Snow
,
Yunyang
Zhang
,
Michael A.
Järvå
,
Janice W.-Y.
Mui
,
Nichollas E.
Scott
,
Eleanor C.
Saunders
,
Runyu
Mao
,
Ruwan
Epa
,
Bruna M.
Da Silva
,
Douglas E. V.
Pires
,
David B.
Ascher
,
Malcolm J.
Mcconville
,
Gideon J.
Davies
,
Spencer J.
Williams
,
Ethan D.
Goddard-Borger
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Catabolism of sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose), the ubiquitous sulfosugar produced by photosynthetic organisms, is an important component of the biogeochemical carbon and sulfur cycles. Here, we describe a pathway for SQ degradation that involves oxidative desulfurization to release sulfite and enable utilization of the entire carbon skeleton of the sugar to support the growth of the plant pathogen Agrobacterium tumefaciens. SQ or its glycoside sulfoquinovosyl glycerol are imported into the cell by an ATP-binding cassette transporter system with an associated SQ binding protein. A sulfoquinovosidase hydrolyzes the SQ glycoside and the liberated SQ is acted on by a flavin mononucleotide-dependent sulfoquinovose monooxygenase, in concert with an NADH-dependent flavin reductase, to release sulfite and 6-oxo-glucose. An NAD(P)H-dependent oxidoreductase reduces the 6-oxo-glucose to glucose, enabling entry into primary metabolic pathways. Structural and biochemical studies provide detailed insights into the recognition of key metabolites by proteins in this pathway. Bioinformatic analyses reveal that the sulfoquinovose monooxygenase pathway is distributed across Alpha- and Betaproteobacteria and is especially prevalent within the Rhizobiales order. This strategy for SQ catabolism is distinct from previously described pathways because it enables the complete utilization of all carbons within SQ by a single organism with concomitant production of inorganic sulfite.
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Jan 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Imine reductases (IREDs) offer biocatalytic routes to chiral amines and have a natural preference for the NADPH cofactor. In previous work, we reported enzyme engineering of the ( R )-selective IRED from Myxococcus stipitatus (( R )-IRED- Ms _V8) yielding a NADH-dependent variant with high catalytic efficiency. However, no IRED with NADH specificity and ( S )-selectivity in asymmetric reductions has yet been reported. Herein, we applied semi-rational enzyme engineering to switch the selectivity of ( R )-IRED- Ms _V8. The quintuple variant A241V/H242Y/N243D/V244Y/A245L showed reverse stereopreference in the reduction of the cyclic imine 2-methylpyrroline compared to the wild-type and afforded the ( S )-amine product with >99% conversion and 91% enantiomeric excess. We also report the crystal-structures of the NADPH-dependent ( R )-IRED- Ms wild-type enzyme and the NADH-dependent ( R )-IRED- Ms _V8 variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant.
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Oct 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Cytochrome P450 CYP153AM.aq from Marinobacter aquaeolei serves as a model enzyme for the terminal (ω-) hydroxylation of medium- to long-chain fatty acids. We have engineered this enzyme using different mutagenesis approaches based on structure-sequence-alignments within the 3DM database and crystal structures of CYP153AM.aq and a homologue CYP153AP.sp. Applying these focused mutagenesis strategies and site-directed saturation mutagenesis, we created a variant that ω-hydroxylates octanoic acid. The M.aqRLT variant exhibited 151-fold improved catalytic efficiency and showed strongly improved substrate binding (25-fold reduced Km compared to the wild type). We then used molecular dynamics simulations to gain deeper insights into the dynamics of the protein. We found the tunnel modifications and the two loop regions showing greatly reduced flexibility in the engineered variant were the main features responsible for stabilizing the enzyme–substrate complex and enhancing the catalytic efficiency. Additionally, we showed that a previously known fatty acid anchor (Q129R) interacts significantly with the ligand to hold it in the reactive position, thereby boosting the activity of the variant M.aqRLT toward octanoic acid. The study demonstrates the significant effects of both substrate stabilization and the impact of enzyme flexibility on catalytic efficiency. These results could guide the future engineering of enzymes with deeply buried active sites to increase or even establish activities toward yet unknown types of substrates.
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Feb 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Mahima
Sharma
,
Palika
Abayakoon
,
Ruwan
Epa
,
Yi
Jin
,
James P.
Lingford
,
Tomohiro
Shimada
,
Masahiro
Nakano
,
Janice W.-Y.
Mui
,
Akira
Ishihama
,
Ethan D.
Goddard-Borger
,
Gideon J.
Davies
,
Spencer J.
Williams
Diamond Proposal Number(s):
[13587, 18598, 24948]
Open Access
Abstract: The sulfosugar sulfoquinovose (SQ) is produced by essentially all photosynthetic organisms on Earth and is metabolized by bacteria through the process of sulfoglycolysis. The sulfoglycolytic Embden–Meyerhof–Parnas pathway metabolizes SQ to produce dihydroxyacetone phosphate and sulfolactaldehyde and is analogous to the classical Embden–Meyerhof–Parnas glycolysis pathway for the metabolism of glucose-6-phosphate, though the former only provides one C3 fragment to central metabolism, with excretion of the other C3 fragment as dihydroxypropanesulfonate. Here, we report a comprehensive structural and biochemical analysis of the three core steps of sulfoglycolysis catalyzed by SQ isomerase, sulfofructose (SF) kinase, and sulfofructose-1-phosphate (SFP) aldolase. Our data show that despite the superficial similarity of this pathway to glycolysis, the sulfoglycolytic enzymes are specific for SQ metabolites and are not catalytically active on related metabolites from glycolytic pathways. This observation is rationalized by three-dimensional structures of each enzyme, which reveal the presence of conserved sulfonate binding pockets. We show that SQ isomerase acts preferentially on the β-anomer of SQ and reversibly produces both SF and sulforhamnose (SR), a previously unknown sugar that acts as a derepressor for the transcriptional repressor CsqR that regulates SQ-utilization. We also demonstrate that SF kinase is a key regulatory enzyme for the pathway that experiences complex modulation by the metabolites SQ, SLA, AMP, ADP, ATP, F6P, FBP, PEP, DHAP, and citrate, and we show that SFP aldolase reversibly synthesizes SFP. This body of work provides fresh insights into the mechanism, specificity, and regulation of sulfoglycolysis and has important implications for understanding how this biochemistry interfaces with central metabolism in prokaryotes to process this major repository of biogeochemical sulfur.
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Feb 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9948, 13587]
Open Access
Abstract: Chiral primary amines are important intermediates in the synthesis of pharmaceutical compounds. Fungal reductive aminases (RedAms) are NADPH-dependent dehydrogenases that catalyse reductive amination of a range of ketones with short-chain primary amines supplied in an equimolar ratio to give corresponding secondary amines. Herein we describe structural and biochemical characterisation as well as synthetic applications of two RedAms from Neosartorya spp. (NfRedAm and NfisRedAm) that display a distinctive activity amongst fungal RedAms, namely a superior ability to use ammonia as the amine partner. Using these enzymes, we demonstrate the synthesis of a broad range of primary amines, with conversions up to >97% and excellent enantiomeric excess. Temperature dependent studies showed that these homologues also possess greater thermal stability compared to other enzymes within this family. Their synthetic applicability is further demonstrated by the production of several primary and secondary amines with turnover numbers (TN) up to 14[thin space (1/6-em)]000 as well as continous flow reactions, obtaining chiral amines such as (R)-2-aminohexane in space time yields up to 8.1 g L−1 h−1. The remarkable features of NfRedAm and NfisRedAm highlight their potential for wider synthetic application as well as expanding the biocatalytic toolbox available for chiral amine synthesis.
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May 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: The Pictet–Spengler reaction is a valuable route to 1,2,3,4-tetrahydro-β-carboline (THBC) and isoquinoline scaffolds found in many important pharmaceuticals. Strictosidine synthase (STR) catalyzes the Pictet–Spengler condensation of tryptamine and the aldehyde secologanin to give (S)-strictosidine as a key intermediate in indole alkaloid biosynthesis. STRs also accept short-chain aliphatic aldehydes to give enantioenriched alkaloid products with up to 99% ee STRs are thus valuable asymmetric organocatalysts for applications in organic synthesis. The STR catalysis of reactions of small aldehydes gives an unexpected switch in stereopreference, leading to formation of the (R)-products. Here we report a rationale for the formation of the (R)-configured products by the STR enzyme from Ophiorrhiza pumila (OpSTR) using a combination of X-ray crystallography, mutational, and molecular dynamics (MD) studies. We discovered that short-chain aldehydes bind in an inverted fashion compared to secologanin leading to the inverted stereopreference for the observed (R)-product in those cases. The study demonstrates that the same catalyst can have two different productive binding modes for one substrate but give different absolute configuration of the products by binding the aldehyde substrate differently. These results will guide future engineering of STRs and related enzymes for biocatalytic applications.
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Jan 2020
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