Engineering a flexible loop in S-adenosyl-L-methionine synthetase enables production of SAM nucleobase analogues with selective biochemical and cellular activity
S-adenosyl-L-methionine (SAM), an essential cofactor in all forms of life, is synthesized by the enzyme methionine adenosyltransferase (MAT) from methionine and ATP. The adenine moiety in SAM appears to have no direct function in catalysis, and some MAT homologs can utilize natural nucleotide triphosphates in vitro, producing the corresponding SAM nucleobase analogues. However, the molecular determinants of nucleotide choice of the MAT enzyme and the cellular significance of the nucleobase in SAM are unclear. In this study, using structure- and bioinformatics-guided mutagenesis, we identify a flexible active-site loop as a major determinant of nucleotide specificity in MAT. Loop mutations and loop swaps convert ATP-selective Escherichia coli MAT into variants that accept GTP, CTP, and UTP, enabling enzymatic synthesis and purification of S-guanosyl-, S-cytosyl-, and S-uracyl-L-methionine. Further, we show that these analogues partially rescue the growth of an E. coli SAM auxotroph under SAM-limited growth conditions. Biochemical assays show that the analogues bind the tested SAM-utilizing enzymes; they serve as substrates for E. coli SAM decarboxylase but do not support detectable methyl transfer by E. coli DNA adenine methyltransferase. These results establish the flexible loop as a gatekeeper of MAT nucleotide specificity and show that this loop can be engineered to produce SAM analogues which can selectively participate in downstream cellular metabolism. Graphical Abstract/ Table of contents only
Nucleoside natural products exhibit diverse chemical architectures and potent biological activities, yet the biosynthetic strategies that generate their structural diversity remain incompletely understood. Here, we elucidate the early stage biosynthetic pathway of the cytidyl-cyclitol natural product K-563 and its derivatives. The cyclitol component is generated by the myo-inositol-1-phosphate synthase (MIPS) family enzyme KesM and is subsequently coupled to the cytidine moiety by KesL using cytidine 5'-triphosphate (CTP), followed by further modifications catalyzed by the phosphatase KesI and the unique dehydrogenase complex KesJ/KesK. The X-ray crystal structure and mutagenesis analyses reveal that KesL adopts the canonical fold of S-adenosyl-l-methionine (SAM) synthetases, which catalyze the adenosylation of l-methionine with adenosine 5'-triphosphate (ATP) in primary metabolism, while the KesL active site is extensively remodeled to accept the cyclitol phosphate with CTP as an atypical substrate pair, thereby generating the cytidyl-cyclitol core structure. This work not only expands the chemical logic of nucleoside biosynthesis but also demonstrates how the SAM synthetase-like protein scaffold is repurposed to catalyze an unusual nucleoside transfer reaction for specialized secondary metabolite assembly.
Zhongtian Yu, Wenhe Zhang, Richiro Ushimaru et al.· Journal of the American Chem...· 0 citations
ATP-grasp enzymes, such as l-amino acid ligases (Lals), catalyze the coupling of carboxylate and amino acid acyl donor substrates to amino acid acyl acceptor substrates via an acyl phosphate intermediate. In addition to their roles in the biosynthesis of natural products, ATP-grasp enzymes have generated significant interest as preparative biocatalysts for the selective synthesis of peptides and amides. TabS is a Lal from Pseudomonas syringae NBRC14081 that couples l-threonine and tabtoxinine-β-lactam to give tabtoxin, which, when hydrolyzed, yields tabtoxinine-β-lactam as a glutamine synthetase inhibitor. However, TabS also has a broad substrate specificity encompassing the coupling of many pairs of l-amino acids, including l-leucine and l-phenylalanine, to form a range of homo- and heterodipeptides. Here we report the structure of TabS, in an apo- form and also in complex with the noncleavable ATP analog AMP-PNP, refined to resolutions of 2.17 and 2.90 Å, respectively. The structures shed light on the broad substrate specificity of TabS and have also informed the exploration, through modeling, mutation, and activity assays, of the possibilities of engineering these enzymes for the coupling of simple carboxylates and amines to form nonpeptide amide products. Rational mutation of hydrophilic residues thought to recognize the amino group of acyl donors and the carboxylate of acyl acceptors has given variants, including S16A/Y44F/Y45F/Y235F/L382F/Q337L/Q339L, which successfully convert the non-amino-acid analogs of the l-leucine donor and acceptor, i.e., 4-methyl pentanoate and 3-methyl butylamine, respectively, to the nonpeptide amide product, where the wild-type TabS displayed negligible activity.
Alex Ascham, Qingyun Tang, Ian J. S. Fairlamb et al.· ACS Catalysis· 0 citations
Strategies for site-specific incorporation of noncanonical amino acids have advanced substantially, enabling access to proteins containing numerous analogues. That noted, many interesting analogues are not α-L-amino acids, and the incorporation of such compounds often requires the development and use of engineered ribosomes. Recently, elongation factor P (EF-P), a translation factor reported to alleviate ribosomal stalling during the incorporation of contiguous L-proline residues, has been shown to facilitate the incorporation of a diverse range of noncanonical amino acids in the presence of native bacterial ribosomes. However, the specific experimental parameters that enable EF-P-mediated enhancement of extensively modified amino acids remain poorly understood. Presently, we examined EF-P-mediated incorporation of four conformationally constrained cyclic dipeptides into position 24 of human RRM1, a nucleic acid recognition motif, using an in vitro transcription-translation system. We compared the effects of ring substitution and conformational isomerism on protein incorporation. Each was incorporated using mono- and bisaminoacylated tRNACUAPhe in comparison to L-phenylalanine. Supplemental EF-P enhanced the incorporation of all analogues significantly, but varied in magnitude substantially between congeners. Bisaminoacylated tRNAs exhibited distinct profiles relative to their monoaminoacylated counterparts. In the absence of exogenous EF-P, bisaminoacylated tRNAs activated with cyclic dipeptides produced similar or lower protein yields relative to their monoaminoacylated counterparts. In contrast, in the absence of added EF-P bisphenylalanyl-tRNA maintained higher incorporation yields than monophenylalanyl-tRNA, as we have observed previously for many aminoacyl-tRNAs containing α-L-amino acids. In the presence of exogenous EF-P, tRNAs activated with two cyclic dipeptides exhibited disproportionately greater yield enhancement, surpassing the corresponding monoaminoacylated analogues. These findings demonstrate that exogenous EF-P-mediated enhancement is influenced by changes within the local structure of the analogues. Additionally, bisaminoacylated tRNAs have the potential of affording significantly greater protein yields, especially in the presence of supplementary EF-P.
Shadow B Harmon, Omar M. Khdour, L. Dedkova et al.· ACS Chemical Biology· 0 citations
S-Adenosyl-l-homocysteine hydrolase (SAHH) reversibly cleaves S-adenosyl-l-homocysteine (SAH) into adenosine and l-homocysteine, regulating the cellular potential to perform S-adenosyl-l-methionine (SAM)-dependent methylation of DNA, RNA, proteins, and small molecules. Another route of SAH metabolism involves the deamination to S-inosyl-l-homocysteine (SIH) followed by degradation to l-homocysteine and inosine catalyzed by S-inosyl-l-homocysteine hydrolase (SIHH). SAHHs have long been studied as regulators of cellular methylation potential, making them attractive targets for antimicrobial, antiviral, and anticancer drug development. In this study, we elucidate the often-reported co-detection of the nucleobases adenine and hypoxanthine in acid-quenched in vitro experiments testing SAHHs and SIHHs. This finding picks up on a reaction mechanism previously established for adenosine and demonstrates that it can also be applied to inosine. Intermediates formed during a dehydratase-like reaction catalyzed by SAHHs and SIHHs were detected by LC-MS/MS. Isolation of these intermediates enabled structural analysis by NMR spectroscopy and their identification as 4′,5′-dehydro nucleosides, as well as systematic assessment of their stability toward pH‑dependent depurination. The dehydratase-like reactivity of SAHHs and SIHHs can be repurposed and utilized in scaled-up reactions to efficiently produce 4′,5′-dehydro adenosine and 4′,5′-dehydro inosine. In addition, non-native nucleoside substrates were converted to the respective 4′,5′-dehydro nucleoside analogues. We propose that the SAHH- and SIHH-catalyzed dehydration reaction offers a straightforward strategy for the enzymatic synthesis of diverse 4′,5′-dehydro nucleosides as valuable precursors for chemical functionalization yielding 4′- or 5′-substituted nucleoside drug candidates.
Lars-Hendrik Koeppl, Philipp Germer, Tim-Luca Wolff et al.· ACS Catalysis· 0 citations
Many plant species accumulate proline as a mechanism to withstand various abiotic stress conditions. Proline synthesis proceeds from either glutamate or ornithine, but in both pathways the last step is catalysed by a δ1-pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.12) showing substrate ambiguity. However, the use of NADH or NADPH implies different turnover number and post-translational regulation: physiological levels of free proline feed-back inhibit only the NADH-dependent activity, whereas Cl- anions in the 20 to 200 mM range stimulate the catalytic rate only if NADPH is the hydride donor. To elucidate the molecular basis of such differences we focused on Ser238 of Arabidopsis thaliana P5C reductase, a highly conserved amino acid residue that is involved in P5C binding and had been shown to form a hydrogen bond with aminomethylene-bisphosphonic acids, leading to enzyme inhibition. Site-directed mutagenesis allowed to obtain a mutated form of the enzyme in which Ser238 has been replaced with Ala. The p.Ser238Ala P5C reductase has been expressed in E. coli, affinity purified and thoroughly characterized. Results showed the abolishment of most differences when using either nicotinamide adenine dinucleotide as the cosubstrate. Data represent a first step toward protein engineering aiming at modulating proline biosynthesis under stress.
G. Forlani, Alessandro Martucci· Plant physiology and biochem...· 0 citations
Electrostatic interactions between arginines and phosphates are central to numerous biological processes. Here, using an integrated approach combining mutagenesis, activity measurements, molecular dynamics (MD) simulations, and NMR, we demonstrate that arginines present in the C-terminal domain of Biotin protein ligase (BPL) are critical for biotinyl-5'-AMP formation. Using NMR-based assays and a group I BPL from Leishmania major (LmBPL), we selectively monitored the first biotinylation step, i.e., formation of biotinyl-5'-AMP from biotin and ATP. The distinct chemical shifts of ATP and AMP enabled us to quantitatively measure the amount of biotinyl-5'-AMP formed by the wild-type enzyme and a C-terminal domain deletion mutant. The mutant displayed remarkably low biotinyl-5'AMP formation compared to the wild-type enzyme. MD simulations of the apo- and ATP-bound forms of LmBPL further identified key interactions between the C-terminal domain arginines (R224, R229) and the γ-phosphate of ATP. The in silico predictions were validated by biochemical studies using R224A, R229A, and R224A/R229A mutants, which displayed remarkably lower biotinyl-5'-AMP formation compared to the wild-type enzyme. Using pyrophosphate as a γ-phosphate mimic, and 31P NMR as a probe, we demonstrate pyrophosphate binding to the wild-type LmBPL but not to the arginine mutants. Consistent with this, biotinyl-5'-AMP formation was completely inhibited by preincubation with pyrophosphate. Taken together, our findings establish a critical role for the C-terminal domain arginines in recognizing ATP phosphates during biotinylation. Extrapolating these findings to other group I and bifunctional group II BPLs, our study reveals a broadly conserved role for the C-terminal domain arginines in regulating biotinylation across the BPL family.
S. Bhatnagar, Debodyuti Sadhukhan, Manoj Kumar Rajak et al.· Journal of Structural Biolog...· 0 citations
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