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Biochemistry. Biokhimiia May 2023O-acetylhomoserine sulfhydrylase is one of the key enzymes in biosynthesis of methionine in Clostridioides difficile. The mechanism of γ-substitution reaction of...
O-acetylhomoserine sulfhydrylase is one of the key enzymes in biosynthesis of methionine in Clostridioides difficile. The mechanism of γ-substitution reaction of O-acetyl-L-homoserine catalyzed by this enzyme is the least studied among the pyridoxal-5'-phosphate-dependent enzymes involved in metabolism of cysteine and methionine. To clarify the role of active site residues Tyr52 and Tyr107, four mutant forms of the enzyme with replacements of these residues with phenylalanine and alanine were generated. Catalytic and spectral properties of the mutant forms were investigated. The rate of γ-substitution reaction catalyzed by the mutant forms with replaced Tyr52 residue decreased by more than three orders of magnitude compared to the wild-type enzyme. The Tyr107Phe and Tyr107Ala mutant forms practically did not catalyze this reaction. Replacements of the Tyr52 and Tyr107 residues led to the decrease in affinity of apoenzyme to coenzyme by three orders of magnitude and changes in the ionic state of the internal aldimine of the enzyme. The obtained results allowed us to assume that Tyr52 is involved in ensuring optimal position of the catalytic coenzyme-binding lysine residue at the stages of C-α-proton elimination and elimination of the side group of the substrate. Tyr107 could act as a general acid catalyst at the stage of acetate elimination.
Topics: Clostridioides difficile; Cysteine Synthase; Catalytic Domain; Clostridioides; Tyrosine; Pyridoxal Phosphate; Methionine; Kinetics
PubMed: 37331706
DOI: 10.1134/S0006297923050036 -
Antimicrobial Agents and Chemotherapy Jul 2023KPC-2 is one of the most relevant serine-carbapenemases among the carbapenem-resistant We previously isolated from the environmental species Chromobacterium...
KPC-2 is one of the most relevant serine-carbapenemases among the carbapenem-resistant We previously isolated from the environmental species Chromobacterium haemolyticum a class A CRH-1 β-lactamase displaying 69% amino acid sequence identity with KPC-2. The objective of this study was to analyze the kinetic behavior and crystallographic structure of this β-lactamase. Our results showed that CRH-1 can hydrolyze penicillins, cephalosporins (except ceftazidime), and carbapenems with similar efficacy compared to KPC-2. Inhibition kinetics showed that CRH-1 is not well inhibited by clavulanic acid, in contrast to efficient inhibition by avibactam (AVI). The high-resolution crystal of the apoenzyme showed that CRH-1 has a similar folding compared to other class A β-lactamases. The CRH-1/AVI complex showed that AVI adopts a chair conformation, stabilized by hydrogen bonds to Ser70, Ser237, Asn132, and Thr235. Our findings highlight the biochemical and structural similarities of CRH-1 and KPC-2 and the potential clinical impact of this carbapenemase in the event of recruitment by pathogenic bacterial species.
Topics: Escherichia coli; Bacterial Proteins; beta-Lactamases; Ceftazidime; Carbapenems; Microbial Sensitivity Tests; Azabicyclo Compounds; Anti-Bacterial Agents; Klebsiella pneumoniae; Drug Combinations
PubMed: 37272821
DOI: 10.1128/aac.00061-23 -
The Journal of Biological Chemistry May 2023CTX-M β-lactamases are a widespread source of resistance to β-lactam antibiotics in Gram-negative bacteria. These enzymes readily hydrolyze penicillins and...
CTX-M β-lactamases are a widespread source of resistance to β-lactam antibiotics in Gram-negative bacteria. These enzymes readily hydrolyze penicillins and cephalosporins, including oxyimino-cephalosporins such as cefotaxime. To investigate the preference of CTX-M enzymes for cephalosporins, we examined eleven active-site residues in the CTX-M-14 β-lactamase model system by alanine mutagenesis to assess the contribution of the residues to catalysis and specificity for the hydrolysis of the penicillin, ampicillin, and the cephalosporins cephalothin and cefotaxime. Key active site residues for class A β-lactamases, including Lys73, Ser130, Asn132, Lys234, Thr216, and Thr235, contribute significantly to substrate binding and catalysis of penicillin and cephalosporin substrates in that alanine substitutions decrease both k and k/K. A second group of residues, including Asn104, Tyr105, Asn106, Thr215, and Thr216, contribute only to substrate binding, with the substitutions decreasing only k/K. Importantly, calculating the average effect of a substitution across the 11 active-site residues shows that the most significant impact is on cefotaxime hydrolysis while ampicillin hydrolysis is least affected, suggesting the active site is highly optimized for cefotaxime catalysis. Furthermore, we determined X-ray crystal structures for the apo-enzymes of the mutants N106A, S130A, N132A, N170A, T215A, and T235A. Surprisingly, in the structures of some mutants, particularly N106A and T235A, the changes in structure propagate from the site of substitution to other regions of the active site, suggesting that the impact of substitutions is due to more widespread changes in structure and illustrating the interconnected nature of the active site.
Topics: Ampicillin; beta-Lactamases; Catalysis; Catalytic Domain; Cefotaxime; Cephalosporins; Drug Resistance; Escherichia coli; Mutagenesis; Penicillins; beta-Lactams; Models, Molecular; Protein Structure, Tertiary
PubMed: 36963495
DOI: 10.1016/j.jbc.2023.104630 -
Journal of Chemical Information and... Feb 2023The catalytic function of lysyl hydroxylase-2 (LH2), a member of the Fe(II)/αKG-dependent oxygenase superfamily, is to catalyze the hydroxylation of lysine to...
The catalytic function of lysyl hydroxylase-2 (LH2), a member of the Fe(II)/αKG-dependent oxygenase superfamily, is to catalyze the hydroxylation of lysine to hydroxylysine in collagen, resulting in stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs). Reports show that high amounts of LH2 lead to the accumulation of HLCCs, causing fibrosis and specific types of cancer metastasis. Some members of the Fe(II)/αKG-dependent family have also been reported to have intramolecular O tunnels, which aid in transporting one of the required cosubstrates into the active site. While LH2 can be a promising target to combat these diseases, efficacious inhibitors are still lacking. We have used computational simulations to investigate a series of 44 small molecules as lead compounds for LH2 inhibition. Tunneling analyses indicate the existence of several intramolecular tunnels. The lengths of the calculated O-transporting tunnels in holoenzymes are relatively longer than those in the apoenzyme, suggesting that the ligands may affect the enzyme's structure and possibly block (at least partially) the tunnels. The sequence alignment analysis between LH enzymes from different organisms shows that all of the amino acid residues with the highest occurrence rate in the oxygen tunnels are conserved. Our results suggest that the enolate form of diketone compounds establishes stronger interactions with the Fe(II) in the active site. Branching the enolate compounds with functional groups such as phenyl and pyridinyl enhances the interaction with various residues around the active site. Our results provide information about possible leads for further LH2 inhibition design and development.
Topics: Collagen; Ferrous Compounds; Hydroxylysine; Lysine; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase
PubMed: 36779232
DOI: 10.1021/acs.jcim.2c01448 -
ACS Infectious Diseases Mar 2023() lacking functional homoserine transacetylase (HTA) is compromised in methionine biosynthesis, protein synthesis, and in the activity of multiple essential...
() lacking functional homoserine transacetylase (HTA) is compromised in methionine biosynthesis, protein synthesis, and in the activity of multiple essential -adenosyl-l-methionine-dependent enzymes. Additionally, deficient mutants are further disarmed by the toxic accumulation of lysine due to a redirection of the metabolic flux toward the lysine biosynthetic pathway. Studies with deletion mutants and crystallographic studies of the apoenzyme have, respectively, validated HTA as an essential enzyme and revealed a ligandable binding site. Seeking a mechanistic characterization of this enzyme, we report crucial structural details and comprehensive functional characterization of HTA. Crystallographic and mass spectral observation of the acetylated HTA intermediate and initial velocity studies were consistent with a ping-pong kinetic mechanism. Wild-type HTA and its site-directed mutants were kinetically characterized with a panel of natural and alternative substrates to understand substrate specificity and identify critical residues for catalysis. Titration experiments using fluorescence quenching showed that both substrates─acetyl-CoA and l-homoserine─engage in a strong and weak binding interaction with HTA. Additionally, substrate inhibition by acetyl-CoA and product inhibition by CoA and -acetyl-l-homoserine were proposed to form the basis of a feedback regulation mechanism. By furnishing key mechanistic and structural information, these studies provide a foundation for structure-based design efforts around this attractive target.
Topics: Mycobacterium tuberculosis; Lysine; Acetyltransferases; Methionine; Acetyl Coenzyme A
PubMed: 36753622
DOI: 10.1021/acsinfecdis.2c00541 -
Archives of Biochemistry and Biophysics Jan 2023Aldehyde-deformylating oxygenase (ADO) is a non-heme di-iron enzyme that catalyzes the deformylation of aldehydes to generate alkanes/alkenes. In this study, we report...
Aldehyde-deformylating oxygenase (ADO) is a non-heme di-iron enzyme that catalyzes the deformylation of aldehydes to generate alkanes/alkenes. In this study, we report for the first time that under anaerobic or limited oxygen conditions, Prochlorococcus marinus (PmADO) can generate full-length fatty alcohols from fatty aldehydes without eliminating a carbon unit. In contrast to ADO's native activity, which requires electrons from the Fd/FNR electron transfer complex, ADO's aldehyde reduction activity requires only NAD(P)H. Our results demonstrated that the yield of alcohol products could be affected by oxygen concentration and the type of aldehyde. Under strictly anaerobic conditions, yields of octanol were up to 31%. Moreover, metal cofactors are not involved in the aldehyde reductase activity of PmADO because the yields of alcohols obtained from apoenzyme and holoenzyme treated with various metals were similar under anaerobic conditions. In addition, PmADO prefers medium-chain aldehydes, specifically octanal (k/K around 15 × 10 μMmin). The findings herein highlight a new activity of PmADO, which may be applied as a biocatalyst for the industrial synthesis of fatty alcohols.
Topics: Aldehyde Reductase; Fatty Alcohols; Cyanobacteria; Oxygenases; Aldehydes; Oxygen
PubMed: 36572346
DOI: 10.1016/j.abb.2022.109498 -
Proceedings of the National Academy of... Nov 2022The DEAH/RHA helicase Prp43 remodels protein-RNA complexes during pre-messenger RNA (mRNA) splicing and ribosome biogenesis. The helicase activity and ATP turnover are...
The DEAH/RHA helicase Prp43 remodels protein-RNA complexes during pre-messenger RNA (mRNA) splicing and ribosome biogenesis. The helicase activity and ATP turnover are intrinsically low and become activated by G-patch (gp) factors in the specific cellular context. The gp motif connects the helicase core to the flexible C-terminal domains, but it is unclear how this affects RecA domain movement during catalysis and the unwinding of RNA substrates. We developed single-molecule Förster Resonance Energy Transfer (smFRET) reporters to study RecA domain movements within Prp43 in real time. Without Pfa1(gp), the domains approach each other adopting predominantly a closed conformation. The addition of Pfa1(gp) induces an open state, which becomes even more prevalent during interaction with RNA. In the open state, Prp43 has reduced contacts with bound nucleotide and shows rapid adenosine diphosphate (ADP) release accelerating the transition from the weak (ADP) to the strong (apo) RNA binding state. Using smFRET labels on the RNA to probe substrate binding and unwinding, we demonstrate that Pfa1(gp) enables Prp43(ADP) to switch between RNA-bound and RNA-unbound states instead of dissociating from the RNA. ATP binding to the apo-enzyme induces the translocation along the RNA, generating the unwinding force required to melt proximal RNA structures. During ATP turnover, Pfa1(gp) stimulates alternating of the RecA domains between open and closed states. Consequently, the translocation becomes faster than dissociation from the substrate in the ADP state, allowing processive movement along the RNA. We provide a mechanistic model of DEAH/RHA helicase motility and reveal the principles of Prp43 regulation by G-patch proteins.
Topics: DEAD-box RNA Helicases; DNA Helicases; RNA; Adenosine Diphosphate; Adenosine Triphosphate
PubMed: 36409901
DOI: 10.1073/pnas.2203567119 -
International Journal of Tryptophan... 2022Major species differences in tryptophan (Trp) metabolism and disposition exist with important physiological, functional and toxicity implications. Unlike mammalian and... (Review)
Review
Major species differences in tryptophan (Trp) metabolism and disposition exist with important physiological, functional and toxicity implications. Unlike mammalian and other species in which plasma Trp exists largely bound to albumin, teleosts and other aquatic species possess little or no albumin, such that Trp entry into their tissues is not hampered, neither is that of environmental chemicals and toxins, hence the need for strict measures to safeguard their aquatic environments. In species sensitive to toxicity of excess Trp, hepatic Trp 2,3-dioxygenase (TDO) lacks the free apoenzyme and its glucocorticoid induction mechanism. These species, which are largely herbivorous, however, dispose of Trp more rapidly and their TDO is activated by smaller doses of Trp than Trp-tolerant species. In general, sensitive species may possess a higher indoleamine 2,3-dioxygenase (IDO) activity which equips them to resist immune insults up to a point. Of the enzymes of the kynurenine pathway beyond TDO and IDO, 2-amino-3-carboxymuconic acid-6-semialdehyde decarboxylase (ACMSD) determines the extent of progress of the pathway towards NAD synthesis and its activity varies across species, with the domestic cat () being the leading species possessing the highest activity, hence its inability to utilise Trp for NAD synthesis. The paucity of current knowledge of Trp metabolism and disposition in wild carnivores, invertebrates and many other animal species described here underscores the need for further studies of the physiology of these species and its interaction with Trp metabolism.
PubMed: 36325027
DOI: 10.1177/11786469221122511 -
Applied Microbiology and Biotechnology Dec 20225-Ketogluconate (5KGA) is a precursor for synthesizing tartrate, a valuable compound used in several industries. In a previous study, Gluconobacter japonicus NBRC 3271...
5-Ketogluconate (5KGA) is a precursor for synthesizing tartrate, a valuable compound used in several industries. In a previous study, Gluconobacter japonicus NBRC 3271 mutant strain D2, which lacks two membranous gluconate 2-dehydrogenases, was shown to produce 5KGA but not 2-ketogluconate from a mixture of glucose and gluconate. In this study, we aimed to develop an efficient 5KGA production system using G. japonicus D2 as the parental strain. D2 produced 5KGA from glucose in a jar fermentor culture; however, 5KGA levels were reduced during the late phase of cultivation. To increase the potential of D2 for 5KGA production, the cytoplasmic metabolism related to the utilization of 5KGA and gluconate was modified; the gno and gntK genes encoding 5KGA reductase and gluconokinase, respectively, were deleted from D2, generating D4. Improved 5KGA production was observed in D4 compared to that in D2, but a significant amount of gluconate remained at the end of cultivation, leading to an unsatisfied yield of 0.83 mol (mol glucose). The conversion of gluconate to 5KGA is catalyzed by pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase (GLDH), which easily forms an apoenzyme by releasing PQQ and calcium ions. Thus, the effects of CaCl addition to the culture medium on 5KGA production by D4 were investigated. We demonstrated that 1 mM CaCl addition positively affected the maintenance of the PQQ-GLDH activity toward gluconate and consequently enhanced 5KGA production, and the yield reached 0.97 mol (mol glucose). KEY POINTS: • An efficient 5KGA production system was developed with Gluconobacter japonicus. • Deleting the gno and gntK genes blocked the catabolism of 5KGA and gluconate. • The addition of 1 mM CaCl efficiently improved the conversion of glucose to 5KGA.
Topics: Calcium Chloride; Gluconobacter; Gluconates; PQQ Cofactor; Glucose
PubMed: 36271931
DOI: 10.1007/s00253-022-12242-0 -
European Journal of Medicinal Chemistry Dec 2022Malaria is caused by the parasite Plasmodium falciparum, which contains an essential non-photosynthetic plastid called the apicoplast. A single DNA polymerase, apPOL, is...
Malaria is caused by the parasite Plasmodium falciparum, which contains an essential non-photosynthetic plastid called the apicoplast. A single DNA polymerase, apPOL, is targeted to the apicoplast, where it replicates and repairs the genome. apPOL has no direct orthologs in mammals and is considered a promising drug target for the treatment and/or prevention of malaria. We previously reported screening the Malaria Box to identify MMV666123 as an inhibitor of apPOL. Herein we extend our studies and report structure-activity relationships for MMV666123 and identify key structural motifs necessary for inhibition. Although attempts to crystallize apPOL with the inhibitor were not fruitful, kinetic analysis and crystal structure determinations of WT and mutant apo-enzymes, facilitated model building and provided insights into the putative inhibitor binding site. Our results validate apPOL as an antimalarial target and provide an avenue for the design of high potency, specific inhibitors of apPOL and other A-family DNA polymerases.
Topics: Animals; Apicoplasts; Plasmodium falciparum; Antimalarials; Kinetics; DNA-Directed DNA Polymerase; Malaria; Protozoan Proteins; Mammals
PubMed: 36191407
DOI: 10.1016/j.ejmech.2022.114751