The Crystal Structure of a Bacterial <scp>l</scp>-Arabinonate Dehydratase Contains a [2Fe-2S] Cluster

Rahman, Mohammad Mubinur; Andberg, Martina; Thangaraj, Senthil K.; Parkkinen, Tarja; Penttilä, Merja; Jänis, Janne; Koivula, Anu; Rouvinen, Juha; Hakulinen, Nina

doi:10.1021/acschembio.7b00304

Cited by 26 publications

(65 citation statements)

References 35 publications

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“…Recently, we solved the first representative of the IlvD/EDD family in its holo form, an L-arabinonate dehydratase from Rhizobium leguminosarum bv trifolii ( Rl ArDHT, PDB code: 5J84). This structure revealed the presence of a [2Fe-2S] cluster and a Mg 2+ ion in the active site 27 .…”

Section: Introductionmentioning

confidence: 96%

The crystal structure of D-xylonate dehydratase reveals functional features of enzymes from the Ilv/ED dehydratase family

Rahman

Andberg

Koivula

et al. 2018

Sci Rep

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The Ilv/ED dehydratase protein family includes dihydroxy acid-, gluconate-, 6-phosphogluconate- and pentonate dehydratases. The members of this family are involved in various biosynthetic and carbohydrate metabolic pathways. Here, we describe the first crystal structure of D-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) at 2.7 Å resolution and compare it with other available enzyme structures from the IlvD/EDD protein family. The quaternary structure of CcXyDHT is a tetramer, and each monomer is composed of two domains in which the N-terminal domain forms a binding site for a [2Fe-2S] cluster and a Mg2+ ion. The active site is located at the monomer-monomer interface and contains residues from both the N-terminal recognition helix and the C-terminus of the dimeric counterpart. The active site also contains a conserved Ser490, which probably acts as a base in catalysis. Importantly, the cysteines that participate in the binding and formation of the [2Fe-2S] cluster are not all conserved within the Ilv/ED dehydratase family, which suggests that some members of the IlvD/EDD family may bind different types of [Fe-S] clusters.

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Section: Introductionmentioning

confidence: 96%

The crystal structure of D-xylonate dehydratase reveals functional features of enzymes from the Ilv/ED dehydratase family

Rahman

Andberg

Koivula

et al. 2018

Sci Rep

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“…Bivalent metal ions do not only stabilize the EDD enzymes but were also described to be crucial for catalysis. As deduced from crystal structures from homologous sugar acid dehydratases the bivalent metal ions mostly Mg 2+ stabilize the oxyanion intermediate generated during the catalytic cycle (Rahman et al, 2017(Rahman et al, , 2018. However, due to missing crystal structures the detailed reaction mechanisms of EDD enzymes remains to be elucidated.…”

Section: Discussionmentioning

confidence: 99%

Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus

Krevet

Shen

Bohnen

et al. 2020

Front. Bioeng. Biotechnol.

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The availability of metabolic intermediates is a prerequisite in many fields ranging from basic research, to biotechnological and biomedical applications as well as diagnostics. 2-keto-3-deoxy-6-phosphogluconate (KDPG) is the key intermediate of the Entner-Doudoroff (ED) pathway for sugar degradation and of sugar acid and sugar polymer breakdown in many organisms including human and plant pathogens. However, so far KDPG is hardly available due to missing efficient synthesis routes. We here report the efficient biocatalytic KDPG production through enzymatic dehydration of 6-phosphogluconate (6PG) up to gram scale using the 6PG dehydratase/Entner-Doudoroff dehydratase (EDD) from Caulobacter crescentus (CcEDD). The enzyme was recombinantly produced in Escherichia coli, purified to apparent homogeneity in a simple one-step procedure using nickel ion affinity chromatography, and characterized with respect to molecular and kinetic properties. The homodimeric CcEDD catalyzed the irreversible 6PG dehydration to KDPG with a V max of 61.6 U mg −1 and a K M of 0.3 mM for 6PG. Most importantly, the CcEDD showed sufficient long-term stability and activity to provide the enzyme in amounts and purity required for the efficient downstream synthesis of KDPG. CcEDD completely converted 1 g 6PG and a straight forward purification method yielded 0.81 g of stereochemically pure KDPG corresponding to a final yield of 90% as shown by HPLC-MS and NMR analyses.

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“…In addition, mutations I433V and S189P, from mutant #1 and mutant #2, respectively, are located on opposing lobes that define the putative substrate entrance to the active site ( Supplementary Figure 11c, d ). These lobes are known to undergo a significant conformational change 62 . In one conformation, the lobes seal the active site away from the solvent surrounding the enzyme ( Supplementary Figure 11c ), presumably to protect the 2Fe-2S cluster in the active site from oxidation.…”

Section: Resultsmentioning

confidence: 99%

Genetically encoded biosensors for branched-chain amino acid metabolism to monitor mitochondrial and cytosolic production of isobutanol and isopentanol in yeast

Zhang¹,

Hammer²,

Carrasco-López³

et al. 2020

Preprint

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22Branched-chain amino acid (BCAA) metabolism can be harnessed to produce many valuable 23 chemicals. Among these, isobutanol, which is derived from valine degradation, has received 24 substantial attention due to its promise as an advanced biofuel. While Saccharomyces cerevisiae is 25 the preferred organism for isobutanol production, the lack of isobutanol biosensors in this organism 26 has limited the ability to screen strains at high throughput. Here, we use a transcriptional regulator of 27 BCAA biosynthesis, Leu3p, to develop the first genetically encoded biosensor for isobutanol 28 production in yeast. Small modifications allowed us to redeploy Leu3p in a second biosensor for 29 isopentanol, another BCAA-derived product of interest. Each biosensor is highly specific to 30 isobutanol or isopentanol, respectively, and was used to engineer metabolic enzymes to increase titers. 31The isobutanol biosensor was additionally employed to isolate high-producing strains, and guide the 32 construction and enhancement of mitochondrial and cytosolic isobutanol biosynthetic pathways, 33 including in combination with optogenetic actuators to enhance metabolic flux. These biosensors 34 promise to accelerate the development of enzymes and strains for branched-chain higher alcohol 35 production, and offer a blueprint to develop biosensors for other products derived from BCAA 36 metabolism. 37 38 Key Words: Branched-chain amino acid metabolism biosensor, LEU3, isobutanol biosensor, 39 isopentanol biosensor, metabolic engineering, high-throughput screen, enzyme engineering, 40 mitochondrial and cytosolic isobutanol pathways 41 42 particular, S. cerevisiae benefits from higher tolerance to alcohols than many bacteria 21 . In addition, 60BCHAs are naturally produced in S. cerevisiae as products of BCAA degradation. Accordingly, 61 metabolic pathways for BCHA production have been engineered by rewiring BCAA biosynthesis and 62 the Ehrlich degradation pathway (Supplementary Figure 1). The upstream BCAA biosynthetic 63 pathway is comprised of three mitochondrially localized enzymes: acetolactate synthase (ALS, 64 encoded by ILV2), ketol-acid reductoisomerase (KARI, encoded by ILV5), and dehydroxyacid 65 dehydratase (DHAD, encoded by ILV3) 22 . Ilv2p, Ilv3p, and Ilv5p convert pyruvate to the valine 66 4 precursor a-ketoisovalerate (a-KIV), which can be exported to the cytosol and converted to 67 isobutanol via the downstream BCAA Ehrlich degradation pathway 23 , comprised of a-ketoacid 68 decarboxylases (a-KDCs) and alcohol dehydrogenases (ADHs). Alternatively, a-KIV can be 69 converted to α-ketoisocaproate (a-KIC) by sequential reactions carried out by a-isopropylmalate 70 synthase (encoded by LEU4 and LEU9), isopropylmalate isomerase (encoded by LEU1), and 3-71 isopropylmalate dehydrogenase (encoded by LEU2). The LEU4 gene produces two forms of α-72 isopropylmalate synthase -a short form located in the cytosol, and a long form localized in 73 mitochondria along with Leu9p 22 . The same Ehrlich degradation pathway converts a-KIC to ...

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The Crystal Structure of a Bacterial l-Arabinonate Dehydratase Contains a [2Fe-2S] Cluster

Cited by 26 publications

References 35 publications

The crystal structure of D-xylonate dehydratase reveals functional features of enzymes from the Ilv/ED dehydratase family

The crystal structure of D-xylonate dehydratase reveals functional features of enzymes from the Ilv/ED dehydratase family

Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus

Genetically encoded biosensors for branched-chain amino acid metabolism to monitor mitochondrial and cytosolic production of isobutanol and isopentanol in yeast

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