Catalytic Intermediate Crystal Structures of Cysteine Desulfurase from the Archaeon Thermococcus onnurineus NA1

Ho, Thien-Hoang; Huynh, Kim-Hung; Nguyen, Diem Quynh; Park, Hyunjae; Jung, Kyoungho; Sur, Bookyo; Ahn, Yeh Jin; Shin, Sun; Kang, Lin Woo

doi:10.1155/2017/5395293

Cited by 5 publications

(5 citation statements)

References 33 publications

(45 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To inform our engineering process, we used a homology model of wt-UstD derived from a distantly related cysteine desulfurase (27% identity) 23 , 24 . Six residues in the predicted active site were chosen for saturation mutagenesis, and we used benzaldehyde ( 2a) as a model substrate for directed evolution ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2022

View full text Add to dashboard Cite

Enzymes are renowned for their catalytic e ciency 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 e cient in a whole-cell biocatalysis format, which circumvents the need for enzyme puri cation, 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. Main TextMajor advances have been made in the practical use of enzymes for enantioselective functional group manipulations 9 . For example, enantioselective reduction of ketones or enantiospeci c hydrolysis of racemic esters are now routine in process chemistry. There have also been impressive strides made in enzymatic C-H activation 10 . However, the development of enzymes to form C-C bonds on preparative scale lags far behind traditional synthetic organic methodology 11 .To ll this gap, we were drawn to a recently described pyridoxal phosphate (PLP) dependent enzyme involved in the biosynthesis of Ustiloxin B, an inhibitor of microtubilin polymerization (Fig. 1A) 12 . This enzyme, UstD, decarboxylates the side chain of l-aspartate (1), forming a putative nucleophilic enamine intermediate (Fig. 1B). This enamine then attacks an aliphatic aldehyde appended to a cyclic tetrapeptide, resulting in the formation of a γ-hydroxy amino acid side chain. The loss of CO 2 renders this enantioselective C-C bond forming reaction irreversible. However, the native substrate for UstD is a complex, cyclic peptide, and it was unknown if this enzyme would react promiscuously with alternative substrates; if so, the enzyme would directly produce γ-hydroxy amino acids (Fig. 1B). Such non-standard amino acids (nsAAs) are found in bioactive natur...

show abstract

Section: Resultsmentioning

confidence: 99%

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

et al. 2022

View full text Add to dashboard Cite

show abstract

“…To inform our engineering process, we used a homology model of wt-UstD derived from a distantly related cysteine desulfurase (27% identity) (17,18). Six residues in the predicted active site were chosen for saturation mutagenesis, and we used benzaldehyde (2a) as a model substrate for directed evolution (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Buller¹,

Ellis²,

Campbell³

et al. 2021

Preprint

View full text Add to dashboard Cite

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.

show abstract

“…In the "assembly" stage, a scaffold protein (SufB) receives sulfur and iron from unknown donors and builds Fe-S clusters; in the "transfer" stage, an ATPase (SufC) facilitates the release of Fe-S clusters from the scaffold SufB, and then the Fe-S clusters are transported to target apoproteins via different carrier proteins [70]. This means that the biosynthetic pathways in Bacteria and Eukarya are quite proven (see, for instance, [63,69,70,[74][75][76]). In contrast, the Fe-S biogenesis in Archaea is not yet accurately known [76][77][78], even if a few SUF systems are there distributed [79][80][81].…”

Section: Cysteine Desulfurasementioning

confidence: 99%

“…This means that the biosynthetic pathways in Bacteria and Eukarya are quite proven (see, for instance, [63,69,70,[74][75][76]). In contrast, the Fe-S biogenesis in Archaea is not yet accurately known [76][77][78], even if a few SUF systems are there distributed [79][80][81]. Premised that L-Cysteine desulfurase IscS and scaffold IscU proteins are universally involved in Fe/S cluster synthesis, in Archaea, the cysteine desulfurase provides one ligand for the binding of an Fe-S cluster.…”

Section: Cysteine Desulfurasementioning

confidence: 99%

The Redox Active [2Fe-2S] Clusters: Key-Components of a Plethora of Enzymatic Reactions—Part I: Archaea

Corsini

Zanello

2022

Inorganics

View full text Add to dashboard Cite

The earliest forms of life (i.e., Archaea, Bacteria, and Eukarya) appeared on our planet about ten billion years after its formation. Although Archaea do not seem to possess the multiprotein machinery constituted by the NIF (Nitrogen Fixation), ISC (Iron Sulfur Cluster), SUF (sulfur mobilization) enzymes, typical of Bacteria and Eukarya, some of them are able to encode Fe-S proteins. Here we discussed the multiple enzymatic reactions triggered by the up-to-date structurally characterized members of the archaeal family that require the crucial presence of structurally characterized [2Fe-2S] assemblies, focusing on their biological functions and, when available, on their electrochemical behavior.

show abstract

Catalytic Intermediate Crystal Structures of Cysteine Desulfurase from the Archaeon Thermococcus onnurineus NA1

Cited by 5 publications

References 33 publications

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

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

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

The Redox Active [2Fe-2S] Clusters: Key-Components of a Plethora of Enzymatic Reactions—Part I: Archaea

Contact Info

Product

Resources

About