Catalytic Machinery Research Articles

Amylolytic enzymes - focus on the alpha-amylases from Archae and plants

Amylolytic enzymes represent a group of starch hydrolases and related enzymes that are active towards the α-glycosidic bonds in starch and related poly- and oligosaccharides. The three best known amylolytic enzymes are α-amylase, β-amylase and glucoamylase that, however, differ from each other by their amino acid sequences, three-dimensional structures, reaction mechanisms and catalytic machineries. In the sequence-based classification of all glycoside hydrolases (GHs) they have therefore been classified into the three independent families: GH13 (α-amylases), GH14 (β-amylases) and GH15 (glucoamylases). Some amylolytic enzymes have been placed to the families GH31 and GH57. The family GH13 together with the families GH70 and GH77 constitutes the clan GH-H, well-known as the α-amylase family. It contains more than 6,000 sequences and covers 30 various enzyme specificities sharing the conserved sequence regions, catalytic TIM-barrel fold, retaining reaction mechanism and catalytic triad. Among the GH13 α-amylases, those produced by plants and archaebacteria exhibit common sequence similarities that distinguish them from the α-amylases of the remaining taxonomic sources. Despite the close evolutionary relatedness between the plant and archaeal α-amylases, there are also specific differences that discriminate them from each other. These specific differences could be used in an effort to reveal the sequence-structural features responsible for the high thermostability of the α-amylases from Archaea.

Small Molecule CD38 Inhibitors: Synthesis of 8-Amino-N1-inosine 5'-monophosphate, Analogues and Early Structure-Activity Relationship.

Although a monoclonal antibody targeting the multifunctional ectoenzyme CD38 is an FDA-approved drug, few small molecule inhibitors exist for this enzyme that catalyzes inter alia the formation and metabolism of the N1-ribosylated, Ca2+-mobilizing, second messenger cyclic adenosine 5′-diphosphoribose (cADPR). N1-Inosine 5′-monophosphate (N1-IMP) is a fragment directly related to cADPR. 8-Substituted-N1-IMP derivatives, prepared by degradation of cyclic parent compounds, inhibit CD38-mediated cADPR hydrolysis more efficiently than related cyclic analogues, making them attractive for inhibitor development. We report a total synthesis of the N1-IMP scaffold from adenine and a small initial compound series that facilitated early delineation of structure-activity parameters, with analogues evaluated for inhibition of CD38-mediated hydrolysis of cADPR. The 5′-phosphate group proved essential for useful activity, but substitution of this group by a sulfonamide bioisostere was not fruitful. 8-NH2-N1-IMP is the most potent inhibitor (IC50 = 7.6 μM) and importantly HPLC studies showed this ligand to be cleaved at high CD38 concentrations, confirming its access to the CD38 catalytic machinery and demonstrating the potential of our fragment approach.

Structure of Pseudomonas aeruginosa spermidine dehydrogenase: a polyamine oxidase with a novel heme-binding fold.

The opportunistic pathogen Pseudomonas aeruginosa can utilize polyamines (including putrescine, cadaverine, 4-aminobutyrate, spermidine, and spermine) as its sole source of carbon and nitrogen. Spermidine dehydrogenase (SpdH) is a component of one of the two polyamine utilization pathways identified in P. aeruginosa, but little is known about its structure and function. Here, we report the first crystal structure of SpdH from P. aeruginosa to 1.85 Å resolution. The resulting core structure confirms that SpdH belongs to the polyamine oxidase (PAO) family with flavin-binding and substrate-binding domains. A unique N-terminal extension wraps around the flavin-binding domain of SpdH and is required for heme binding, placing a heme cofactor in close proximity to the FAD cofactor. Structural and mutational analysis reveals that residues in the putative active site at the re side of the FAD isoalloxazine ring form part of the catalytic machinery. PaSpdH features an unusual active site and lacks the conserved lysine that forms part of a lysine-water-flavin N5 atom interaction in other PAO enzymes characterized to date. Mutational analysis further confirms that heme is required for catalytic activity. This work provides an important starting point for understanding the role of SpdH, which occurs universally in P. aeruginosa strains, in polyamine metabolism.

Genomic and in silico protein structural analyses provide insights into marine polysaccharide-degrading enzymes in the sponge-derived Pseudoalteromonas sp. PA2MD11

Active heterotrophic metabolism is a critical metabolic role performed by sponge-associated microorganisms, but little is known about their capacity to metabolize marine polysaccharides (MPs). Here, we investigated the genome of the sponge-derived Pseudoalteromonas sp. strain PA2MD11 focusing on its macroalgal carbohydrate-degrading potential. Carbohydrate-active enzymes (CAZymes) for the depolymerization of agar and alginate were found in PA2MD11's genome, including glycoside hydrolases (GHs) and polysaccharide lyases (PLs) belonging to families GH16, GH50 and GH117, and PL6 and PL17, respectively. A gene potentially encoding a sulfatase was also identified, which may play a role in the strain's ability to consume carrageenans. The complete metabolism of agar and alginate by PA2MD11 could also be predicted and was consistent with the results obtained in physiological assays. The polysaccharide utilization locus (PUL) potentially involved in the metabolism of agarose contained mobile genetic elements from other marine Gammaproteobacteria and its unusual larger size might be due to gene duplication events. Homology modelling and structural protein analyses of the agarases, alginate lyases and sulfatase depicted clear conservation of catalytic machinery and protein folding together with suitable industrially-relevant features. Pseudoalteromonas sp. PA2MD11 is therefore a source of potential MP-degrading biocatalysts for biorefinery applications and in the preparation of pharmacologically-active oligosaccharides.

Catalytic machinery of methane oxidation in particulate methane monooxygenase (pMMO)

In this focused review, we portray the recently reported 2.5 Å cyro-EM structure of the particulate methane monooxygenase (pMMO) from M. capsulatus (Bath). The structure of the functional holo-pMMO near atomic resolution has uncovered the sites of the copper cofactors including the location of the active site in the enzyme. The three coppers seen in the original X-ray crystal structures of the enzyme are now augmented by additional coppers in the transmembrane domain as well as in the water-exposed C-terminal subdomain of the PmoB subunit. The cryo-EM structure offers the first glimpse of the catalytic machinery capable of methane oxidation with high selectivity and efficiency. The findings are entirely consistent with the biochemical and biophysical findings previously reported in the literature, including the chemistry of hydrocarbon hydroxylation, regeneration of the catalyst for multiple turnovers, and the mechanism of aborting non-productive cycles to ensure kinetic competence.

Slow-Onset, Potent Inhibition of Mandelate Racemase by 2-Formylphenylboronic Acid. An Unexpected Adduct Clasps the Catalytic Machinery.

o-Carbonyl arylboronic acids such as 2-formylphenylboronic acid (2-FPBA) are employed in biocompatible conjugation reactions with the resulting iminoboronate adduct stabilized by an intramolecular N-B interaction. However, few studies have utilized these reagents as active site-directed enzyme inhibitors. We show that 2-FPBA is a potent reversible, slow-onset inhibitor of mandelate racemase (MR), an enzyme that has served as a valuable paradigm for understanding enzyme-catalyzed abstraction of an α-proton from a carbon acid substrate with a high pKa. Kinetic analysis of the progress curves for the slow onset of inhibition of wild-type MR using a two-step kinetic mechanism gave Ki and Ki* values of 5.1 ± 1.8 and 0.26 ± 0.08 μM, respectively. Hence, wild-type MR binds 2-FPBA with an affinity that exceeds that for the substrate by ∼3000-fold. K164R MR was inhibited by 2-FPBA, while K166R MR was not inhibited, indicating that Lys 166 was essential for inhibition. Unexpectedly, mass spectrometric analysis of the NaCNBH3-treated enzyme-inhibitor complex did not yield evidence of an iminoboronate adduct. 11B nuclear magnetic resonance spectroscopy of the MR·2-FPBA complex indicated that the boron atom was sp3-hybridized (δ 6.0), consistent with dative bond formation. Surprisingly, X-ray crystallography revealed the formation of an Nζ-B dative bond between Lys 166 and 2-FPBA with intramolecular cyclization to form a benzoxaborole, rather than the expected iminoboronate. Thus, when o-carbonyl arylboronic acid reagents are employed to modify proteins, the structure of the resulting product depends on the protein architecture at the site of modification.

Exploration of the Activation Mechanism of the Epigenetic Regulator MLL3: A QM/MM Study.

The mixed lineage leukemia 3 or MLL3 is the enzyme in charge of the writing of an epigenetic mark through the methylation of lysine 4 from the N-terminal domain of histone 3 and its deregulation has been related to several cancer lines. An interesting feature of this enzyme comes from its regulation mechanism, which involves its binding to an activating dimer before it can be catalytically functional. Once the trimer is formed, the reaction mechanism proceeds through the deprotonation of the lysine followed by the methyl-transfer reaction. Here we present a detailed exploration of the activation mechanism through a QM/MM approach focusing on both steps of the reaction, aiming to provide new insights into the deprotonation process and the role of the catalytic machinery in the methyl-transfer reaction. Our finding suggests that the source of the activation mechanism comes from conformational restriction mediated by the formation of a network of salt-bridges between MLL3 and one of the activating subunits, which restricts and stabilizes the positioning of several residues relevant for the catalysis. New insights into the deprotonation mechanism of lysine are provided, identifying a valine residue as crucial in the positioning of the water molecule in charge of the process. Finally, a tyrosine residue was found to assist the methyl transfer from SAM to the target lysine.

Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins

Prenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible β-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.

Copper Centers in the Cryo-EM Structure of Particulate Methane Monooxygenase Reveal the Catalytic Machinery of Methane Oxidation.

The particulate methane monooxygenase (pMMO) is the first enzyme in the C1 metabolic pathway in methanotrophic bacteria. As this enzyme converts methane into methanol efficiently near room temperature, it has become the paradigm for developing an understanding of this difficult C1 chemistry. pMMO is a membrane-bound protein with three subunits (PmoB, PmoA, and PmoC) and 12-14 coppers distributed among different sites. X-ray crystal structures that have revealed only three mononuclear coppers at three sites have neither disclosed the location of the active site nor the catalytic mechanism of the enzyme. Here we report a cyro-EM structure of holo-pMMO from Methylococcus capsulatus (Bath) at 2.5 Å, and develop quantitative electrostatic-potential profiling to scrutinize the nonprotein densities for signatures of the copper cofactors. Our results confirm a mononuclear CuI at the A site, resolve two CuIs at the B site, and uncover additional CuI clusters at the PmoA/PmoC interface within the membrane (D site) and in the water-exposed C-terminal subdomain of the PmoB (E clusters). These findings complete the minimal set of copper factors required for catalytic turnover of pMMO, offering a glimpse of the catalytic machinery for methane oxidation according to the chemical principles underlying the mechanism proposed earlier.

JMJD6 Is a Druggable Oxygenase That Regulates AR-V7 Expression in Prostate Cancer.

Endocrine resistance (EnR) in advanced prostate cancer is fatal. EnR can be mediated by androgen receptor (AR) splice variants, with AR splice variant 7 (AR-V7) arguably the most clinically important variant. In this study, we determined proteins key to generating AR-V7, validated our findings using clinical samples, and studied splicing regulatory mechanisms in prostate cancer models. Triangulation studies identified JMJD6 as a key regulator of AR-V7, as evidenced by its upregulation with in vitro EnR, its downregulation alongside AR-V7 by bromodomain inhibition, and its identification as a top hit of a targeted siRNA screen of spliceosome-related genes. JMJD6 protein levels increased (P < 0.001) with castration resistance and were associated with higher AR-V7 levels and shorter survival (P = 0.048). JMJD6 knockdown reduced prostate cancer cell growth, AR-V7 levels, and recruitment of U2AF65 to AR pre-mRNA. Mutagenesis studies suggested that JMJD6 activity is key to the generation of AR-V7, with the catalytic machinery residing within a druggable pocket. Taken together, these data highlight the relationship between JMJD6 and AR-V7 in advanced prostate cancer and support further evaluation of JMJD6 as a therapeutic target in this disease. SIGNIFICANCE: This study identifies JMJD6 as being critical for the generation of AR-V7 in prostate cancer, where it may serve as a tractable target for therapeutic intervention.

The catalytic machinery of the FAD-dependent AtBBE-like protein 15 for alcohol oxidation: Y193 and Y479 form a catalytic base, Q438 and R292 an alkoxide binding site

Monolignol oxidoreductases are members of the berberine bridge enzyme–like (BBE-like) protein family (pfam 08031) that oxidize monolignols to the corresponding aldehydes. They are FAD-dependent enzymes that exhibit the para-cresolmethylhydroxylase-topology, also known as vanillyl oxidase-topology. Recently, we have reported the structural and biochemical characterization of two monolignol oxidoreductases from Arabidopsis thaliana, AtBBE13 and AtBBE15. Now, we have conducted a comprehensive site directed mutagenesis study for AtBBE15, to expand our understanding of the catalytic mechanism of this enzyme class. Based on the kinetic properties of active site variants and molecular dynamics simulations, we propose a refined, structure-guided reaction mechanism for the family of monolignol oxidoreductases. Here, we propose that this reaction is facilitated stepwise by the deprotonation of the allylic alcohol and a subsequent hydride transfer from the Cα-atom of the alkoxide to the flavin. We describe an excessive hydrogen bond network that enables the catalytic mechanism of the enzyme. Within this network Tyr479 and Tyr193 act concertedly as active catalytic bases to facilitate the proton abstraction. Lys436 is indirectly involved in the deprotonation as this residue determines the position of Tyr193 via a cation-π interaction. The enzyme forms a hydrophilic cavity to accommodate the alkoxide intermediate and to stabilize the transition state from the alkoxide to the aldehyde. By means of molecular dynamics simulations, we have identified two different and distinct binding modes for the substrate in the alcohol and alkoxide state. The alcohol interacts with Tyr193 and Tyr479 while Arg292, Gln438 and Tyr193 form an alkoxide binding site to accommodate this intermediate. The pH-dependency of the activity of the active site variants revealed that the integrity of the alkoxide binding site is also crucial for the fine tuning of the pKa of Tyr193 and Tyr479. Sequence alignments showed that key residues for the mechanism are highly conserved, indicating that our proposed mechanism is not only relevant for AtBBE15 but for the majority of BBE-like proteins.

CE16 acetylesterases: in silico analysis, catalytic machinery prediction and comparison with related SGNH hydrolases.

The online version contains supplementary material available at 10.1007/s13205-020-02575-w.

Evolution of catalytic machinery: three-component nanorotor catalyzes formation of four-component catalytic machinery.

The three-component nanorotor [Cu2(S)(R)]2+ (k298 = 46.0 kHz) that is a catalyst for a CuAAC reaction binds the click product at each of its copper centers thereby creating a new platform and a dynamic slider-on-deck system. Due to this sliding motion (k298 = 65.0 kHz) the zinc-porphyrin bound N-methylpyrrolidine is efficiently released into solution and catalyzes a follow-up Michael addition.

NleB/SseK-catalyzed arginine-glycosylation and enteropathogen virulence are finely tuned by a single variable position contiguous to the catalytic machinery.

NleB/SseK effectors are arginine-GlcNAc-transferases expressed by enteric bacterial pathogens that modify host cell proteins to disrupt signaling pathways. While the conserved Citrobacter rodentium NleB and E. coli NleB1 proteins display a broad selectivity towards host proteins, Salmonella enterica SseK1, SseK2, and SseK3 have a narrowed protein substrate selectivity. Here, by combining computational and biophysical experiments, we demonstrate that the broad protein substrate selectivity of NleB relies on Tyr284NleB/NleB1, a second-shell residue contiguous to the catalytic machinery. Tyr284NleB/NleB1 is important in coupling protein substrate binding to catalysis. This is exemplified by S286YSseK1 and N302YSseK2 mutants, which become active towards FADD and DR3 death domains, respectively, and whose kinetic properties match those of enterohemorrhagic E. coli NleB1. The integration of these mutants into S. enterica increases S. enterica survival in macrophages, suggesting that better enzymatic kinetic parameters lead to enhanced virulence. Our findings provide insights into how these enzymes finely tune arginine-glycosylation and, in turn, bacterial virulence. In addition, our data show how promiscuous glycosyltransferases preferentially glycosylate specific protein substrates.

Cross β amyloid assemblies as complex catalytic machinery.

How modern enzymes evolved as complex catalytic machineries to facilitate diverse chemical transformations is an open question for the emerging field of systems chemistry. Inspired by Nature's ingenuity in creating complex catalytic structures for exotic functions, short peptide-based cross β amyloid sequences have been shown to access intricate binding surfaces demonstrating the traits of extant enzymes and proteins. Based on their catalytic proficiencies reported recently, these amyloid assemblies have been argued as the earliest protein folds. Herein, we map out the recent progress made by our laboratory and other research groups that demonstrate the catalytic diversity of cross β amyloid assemblies. The important role of morphology and specific mutations in peptide sequences has been underpinned in this review. We have divided the feature article into different sections where examples from biology have been covered demonstrating the mechanism of extant biocatalysts and compared with recent works on cross β amyloid folds showing covalent catalysis, aldolase, hydrolase, peroxidase-like activities and complex cascade catalysis. Beyond equilibrium, we have extended our discussion towards transient catalytic amyloid phases mimicking the energy driven cytoskeleton polymerization. Finally, a future outlook has been provided on the way ahead for short peptide-based systems chemistry approaches that can lead to the development of robust catalytic networks with improved enzyme-like proficiencies and higher complexities. The discussed examples along with the rationale behind selecting specific amino acids sequence will benefit readers to design systems for achieving catalytic reactivity similar to natural complex enzymes.

Tagging of RPS9 as a tool for ribosome purification and identification of ribosome-associated proteins

Ribosomes, the catalytic machinery required for protein synthesis, are comprised of 4 ribosomal RNAs and about 80 ribosomal proteins in mammals. Ribosomes further interact with numerous associated factors that regulate their biogenesis and function. As mutations of ribosomal proteins and ribosome-associated proteins cause many diseases, it is important to develop tools by which ribosomes can be purified efficiently and with high specificity. Here, we designed a method to purify ribosomes from human cell lines by C-terminally tagging human RPS9, a protein of the small ribosomal subunit. The tag consists of a flag peptide and a streptavidin-binding peptide (SBP) separated by the tobacco etch virus (TEV) protease cleavage site. We demonstrate that RPS9-Flag-TEV-SBP (FTS) is efficiently incorporated into the ribosome without interfering with regular protein synthesis. Using HeLa-GFP-G3BP1 cells stably expressing RPS9-FTS or, as a negative control, mCherry-FTS, we show that complete ribosomes as well as numerous ribosome-associated proteins are efficiently and specifically purified following pull-down of RPS9-FTS using streptavidin beads. This tool will be helpful for the characterization of human ribosome heterogeneity, post-translational modifications of ribosomal proteins, and changes in ribosome-associated factors after exposing human cells to different stimuli and conditions.

Structural asymmetry governs the assembly and GTPase activity of McrBC restriction complexes

McrBC complexes are motor-driven nucleases functioning in bacterial self-defense by cleaving foreign DNA. The GTP-specific AAA + protein McrB powers translocation along DNA and its hydrolysis activity is stimulated by its partner nuclease McrC. Here, we report cryo-EM structures of Thermococcus gammatolerans McrB and McrBC, and E. coli McrBC. The McrB hexamers, containing the necessary catalytic machinery for basal GTP hydrolysis, are intrinsically asymmetric. This asymmetry directs McrC binding so that it engages a single active site, where it then uses an arginine/lysine-mediated hydrogen-bonding network to reposition the asparagine in the McrB signature motif for optimal catalytic function. While the two McrBC complexes use different DNA-binding domains, these contribute to the same general GTP-recognition mechanism employed by all G proteins. Asymmetry also induces distinct inter-subunit interactions around the ring, suggesting a coordinated and directional GTP-hydrolysis cycle. Our data provide insights into the conserved molecular mechanisms governing McrB family AAA + motors.

Structure and function of bacteriophage CBA120 ORF211 (TSP2), the determinant of phage specificity towards E. coli O157:H7

The genome of Escherichia coli O157:H7 bacteriophage vB_EcoM_CBA120 encodes four distinct tailspike proteins (TSPs). The four TSPs, TSP1-4, attach to the phage baseplate forming a branched structure. We report the 1.9 Å resolution crystal structure of TSP2 (ORF211), the TSP that confers phage specificity towards E. coli O157:H7. The structure shows that the N-terminal 168 residues involved in TSPs complex assembly are disordered in the absence of partner proteins. The ensuing head domain contains only the first of two fold modules seen in other phage vB_EcoM_CBA120 TSPs. The catalytic site resides in a cleft at the interface between adjacent trimer subunits, where Asp506, Glu568, and Asp571 are located in close proximity. Replacement of Asp506 and Asp571 for alanine residues abolishes enzyme activity, thus identifying the acid/base catalytic machinery. However, activity remains intact when Asp506 and Asp571 are mutated into asparagine residues. Analysis of additional site-directed mutants in the background of the D506N:D571N mutant suggests engagement of an alternative catalytic apparatus comprising Glu568 and Tyr623. Finally, we demonstrate the catalytic role of two interacting glutamate residues of TSP1, located in a cleft between two trimer subunits, Glu456 and Glu483, underscoring the diversity of the catalytic apparatus employed by phage vB_EcoM_CBA120 TSPs.

Hybrid Inorganic-Biological Systems: Faradaic and Quantum Efficiency, Necessary but Not Sufficient

Adam J. Rieth is a postdoctoral scholar in the Nocera group at Harvard University. He completed his undergraduate studies at the University of North Carolina at Chapel Hill, in his hometown. He then earned his Ph.D. in the laboratory of Prof. Mircea Dincă at the Massachusetts Institute of Technology, where he designed metal-organic frameworks for water harvesting. For the past year in the Nocera group, he has investigated the mechanisms and kinetics of photochemical processes that activate strong bonds. Daniel G. Nocera is the Patterson Rockwood Professor of Energy at Harvard University. He created the field of proton-coupled electron transfer (PCET) at a mechanistic level with the first kinetics measurements and theory of PCET. From this platform, he invented the Artificial Leaf and Bionic Leaf for the solar-driven fixation of carbon dioxide from air in the form of biomass and liquid fuels at energy efficiencies that are 10× greater than natural photosynthesis. He has since elaborated the Bionic Leaf for the solar-driven fixation of nitrogen from air for distributed and carbon-negative ammonia (and fertilizer) synthesis.

Rapid computational evaluation of small‐molecule hydrolase mimics for preorganized H‐bond networks

AbstractSpirocyclic oligomers formed by coupling chiral cyclic building blocks carrying the catalytic machinery of hydrolases (spiroligozymes) catalyze the transesterification of vinyl trifluoromethylacetate with methanol but suffer from the lack of enzyme‐like preorganization of their catalytic functionalities in a well‐defined geometry via H‐bond networks. The computational protocol presented herein combines different levels of theories to rapidly explore structural modifications for an improved structural preorganization. Calculations predict that a modification as simple as replacing the five‐membered building block holding the alcohol moiety with a six‐membered analog in spiroligozymes significantly increases the occupancy of the H‐bond between the benzyl alcohol‐pyridine nucleophilic dyad that mimics the Ser‐His‐Asp/Glu triad of hydrolases. The computed energy profile is indicative of faster acylation of this derivative compared to the parent spiroligozyme and highlights the importance of inclusion of an oxyanion hole motif for efficient catalysis.