Gamma cleavage is a rate-determining step in the gamma-elimination reaction of L-methionine analogues catalyzed by methionine-gamma-lyase

Foo, Timothy C; Meacham, Jesse; Terentis, Andrew C.; Venkatachalam, K.V.

doi:10.1016/j.bbapap.2021.140652

Cited by 5 publications

(14 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The identity of the 352 nm band is uncertain, as the VGK intermediate (VI) was previously assigned in the 320-340 nm range. 42,43 The 485 nm band has been previously assigned as vinyl crotonate species (VIII) that forms prior to product release. 42,43 This feature of the spectrum was constant at initial velocities, corresponding to a steady state, while a strong absorbance band grew at ~320 nm corresponding to accumulation of 2 from turnover and a decrease in the 352 nm peak after 30 minutes (Figure 6C).…”

Section: Resultsmentioning

confidence: 99%

Multiplexed deconvolution of enzyme function in a PLP-dependent protein family

Zmich

Perkins

Bingman

et al. 2023

Preprint

View full text Add to dashboard Cite

Sequence-based functional annotation of enzymes is an essential step in the discovery and development of new biocatalysts. The vinylglycine ketimine (VGK) subfamily of pyridoxal-phosphate dependent enzymes plays critical roles in sulfur metabolism and is home to a growing range of secondary metabolic enzymes that synthesize noncanonical amino acids. However, discovery of useful new enzymes has been slowed because functional assignments within the VGK sub-family are convoluted by pervasive mis-annotation. Here, we used a whole-cell substrate multiplexed screening approach to rapidly measure catalytic activities of 40 homologs in the VGK subfamily. This strategy gives direct information on enzyme specificity without having to purify each enzyme or measure individual kinetic constants. We identified a thermostable cystathionine γ-lyase from Thermobifida fusca and performed mechanistic and structural studies. For biocatalytic applications, we identified a well-behaved, thermostable, and promiscuous amino acid γ-synthase from Caldicellulosiruptor hydrothermalis (CahyGS). We showed CahyGS can catalyze a stereoselective γ-addition into L-allylglycine, providing preparative-scale access to a unique set of γ-branched amino acids. High resolution crystal structures of CahyGS show an open-closed transition associated with ligand binding and provide a basis for subsequent engineering. Together, these data show how multiplexed screening approaches aid in the rapid deconvolution of enzyme function and identify enzymes with useful properties for enzymology and biocatalysis.

show abstract

Section: Resultsmentioning

confidence: 99%

Multiplexed deconvolution of enzyme function in a PLP-dependent protein family

Zmich

Perkins

Bingman

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…There is only a single intermediate in the catalytic cycle that could generate this absorbance band, the pre-elimination quinonoid, E(Q) (Figure 1B, blue). 19 Addition of 5 mM OAHS yielded an analogous peak at 500 nm, albeit more slowly. Difference spectra of both substrates further revealed a shoulder at ∼470 nm (which is partially eclipsed by the 500 nm peak) and matched the expected λ max for the E(AC) (Figure S2).…”

Section: Kinetic Characterization Of a Promiscuous γ-Lyasementioning

confidence: 98%

“…Most of these intermediates of VGK enzymes have been characterized and are shared among enzymes in this subfamily (Figure 1C). 7,13,19,25,26 In the absence of substrate, Thf uCGL has a strong absorbance peak at 425 nm, consistent with an internal aldimine, E(Ain) (Figure 1B, black). Upon addition of 5 mM Cth, we observed a new λ max at 352 nm with a strong shoulder at 480 nm (Figure 1B, gray).…”

Section: Kinetic Characterization Of a Promiscuous γ-Lyasementioning

confidence: 99%

“…18 Further, the stereoselectivity of Cβ deprotonation is convoluted as exchange at this position has been reported to be stereoselective in some VGK enzymes 15 but not in others. 19 Regardless, deprotonation forms an enamine that is an intermediate in the E 1 CB elimination across Cβ−Cγ, with the identity of the leaving group serving as a major difference between VGK enzymes. The orientation of the vinyl group of the VGK intermediate can be in either an s-cis or s-trans configuration (Scheme 1 and Figure 1).…”

Section: ■ Introductionmentioning

confidence: 99%

“…Alternatively, there are many catabolic γ-lyases in nature wherein the VGK instead tautomerizes to form an electrophilic amino crotonate intermediate that is subsequently released into solution and hydrolyzed to form ammonia and α-ketobutyrate (αKB). 8,19,20,22 Identifying the determinants between γsynthase and γ-lyase has been challenging, as enzymes in transsulfuration pathways frequently have both synthase and lyase activities. 6,8,9,11,18 To address these questions, we pursued a comprehensive structural and mechanistic study of a Thermobifida fusca cystathionine γ-lyase (Thf uCGL).…”

Section: ■ Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Elucidation of the Stereochemical Mechanism of Cystathionine γ-Lyase Reveals How Substrate Specificity Constrains Catalysis

Zmich,

Perkins,

Bingman

et al. 2024

ACS Catal.

View full text Add to dashboard Cite

Pyridoxal phosphate (PLP)-dependent enzymes play essential roles in metabolism and have found applications for organic synthesis and as enzyme therapeutics. The vinylglycine ketimine (VGK) subfamily hosts a growing set of enzymes that play diverse roles in primary and secondary metabolism. However, the molecular determinants of substrate specificity and the complex acid−base chemistry that enables VGK catalysis remain enigmatic. We use a recently discovered amino acid γ-lyase as a model system to probe catalysis in this enzyme family. We discovered that two stereochemically distinct proton transfer pathways occur. Combined kinetic and spectroscopic analysis revealed that progression through the catalytic cycle is correlated with the presence of an Hbond donor after Cγ of an amino acid substrate, suggesting that substrate binding is kinetically coupled to a conformational change. High-resolution X-ray crystallography shows that cystathionine-γ-lyases generate an s-trans intermediate and that this geometry is likely conserved throughout the VGK family. An H-bond acceptor in the active site templates substrate binding but does so by preorganizing substrates away from catalytically productive orientations. Mutagenesis eliminates this preorganization, such that there is a relaxation of the substrate specificity but an increase in k cat for diverse substrates. We exploit this information to perform a preparative-scale α,β,β-trideuteration of polar amino acids. Together, these data untangle a complex mode of substrate specificity and provide a foundation for the future study and applications of VGK enzymes.

show abstract