Reaction Mechanism and Substrate Specificity of Iso-orotate Decarboxylase: A Combined Theoretical and Experimental Study

The C-C bond cleavage catalyzed by metal-dependent iso-orotate decarboxylase (IDCase) from the thymidine salvage pathway is of interest for the elucidation of a (hypothetical) DNA demethylation pathway. IDCase appears also as a promising candidate for the synthetic regioselective carboxylation of N-heteroaromatics. Herein, we report a joint experimental-theoretical study to gain insights into the metal identity, reaction mechanism, and substrate specificity of IDCase. In contrast to previous assumptions, the enzyme is demonstrated by ICPMS/MS measurements to contain a catalytically relevant Mn2+ rather than Zn2+. Quantum chemical calculations revealed that decarboxylation of the natural substrate (5-carboxyuracil) proceeds via a (reverse) electrophilic aromatic substitution with formation of CO2. The occurrence of previously proposed tetrahedral carboxylate intermediates with concomitant formation of HCO3- could be ruled out on the basis of prohibitively high energy barriers. In contrast to related o-benzoic acid decarboxylases, such as γ-resorcylate decarboxylase and 5-carboxyvanillate decarboxylase, which exhibit a relaxed substrate tolerance for phenolic acids, IDCase shows high substrate fidelity. Structural and energy comparisons suggest that this is caused by a unique hydrogen bonding of the heterocyclic natural substrate (5-carboxyuracil) to the surrounding residues. Analysis of calculated energies also shows that the reverse carboxylation of uracil is impeded by a strongly disfavored uphill reaction

MLL2 Is Required in Oocytes for Bulk Histone 3 Lysine 4 Trimethylation and Transcriptional Silencing

Conditional knockout mouse strategies identify the histone methyltranferase MLL2 as a key player in epigenetic reprogramming of female gametes

Randomized clinical study using xenograft blocks loaded with bone morphogenetic protein‐2 or autogenous bone blocks for ridge augmentation – a three‐dimensional analysis

OBJECTIVES: To test whether or not the use of a xenogeneic block loaded with rhBMP-2 results in superior radiological and profilometric outcomes compared to an autogenous bone block. MATERIALS AND METHODS: Twenty-four patients randomly received a xenogeneic block loaded with rhBMP-2 (test) or an autogenous bone block (control) for primary augmentation. The ridge width (RW) was evaluated by means of a CBCT scan after augmentation surgery and at 4 months, prior to implant placement. Surface scans were taken prior to augmentation and at 4 months for profilometric analyses. Data were analyzed with Wilcoxon-signed rank test, Mann-Whitney test, or nonparametric ANOVA models. RESULTS: The median RW after augmentation amounted to 7.13 mm (Q1 = 6.02; Q3 = 8.47) for test and 6.86 mm (Q1 = 5.99; Q3 = 8.95) for control. During 4 months of healing, the total RW decreased statistically significantly and measured 5.35 mm (Q1 = 4.53; Q3 = 6.7) for test and 5.15 mm (Q1 = 3.57; Q3 = 7.24) for control (p = 0.0005). The differences between the groups were not statistically significant (p > 0.5899). The buccal soft tissue contour slightly increased for test (0.83 mm; Q1 = 0.62; Q3 = 1.87) and control (1.16 mm; Q1 = 0.50; Q3 = 1.44). CONCLUSIONS: Both treatment modalities successfully increased the ridge width to a similar extent. The shrinkage during healing was not greater in the test than in the control group. The impact of hard tissue augmentation on the soft tissue contour was, however, minimal

Primary bone augmentation leads to equally stable marginal tissue conditions comparing the use of xenograft blocks infused with BMP-2 and autogenous bone blocks: A 3D analysis after 3 years

OBJECTIVES To test whether or not primary bone augmentation using xenograft blocks infused with BMP-2 or autogenous bone blocks lead to similar results regarding the implant survival and 3D marginal soft tissue contours. METHODS Twenty-four patients with an insufficient ridge width for implant placement in need of primary augmentation were randomly assigned to either a block of deproteinized bovine bone mineral infused with rhBMP-2 (BMP) or an intraorally harvested block of autogenous bone (ABB). At 4 months, 1-4 dental implants were placed in the regenerated area. After crown insertion and at 3 years, peri-implant tissue parameters, two- and three-dimensional radiographic parameters, and soft tissue contour changes were evaluated. Explorative mixed model analyses were performed. The level of significance was set at 5%. RESULTS At the 3-year follow-up, 23 patients with 40 implants were evaluated. The implant survival rate was 100% in both groups. At baseline, the marginal hard tissue levels amounted to -0.4 ± 0.8 mm (mean ± standard deviation) in the BMP group and -0.7 ± 1.0 mm in the ABB group. At 3 years, these values were -0.2 ± 0.4 mm (BMP) and -0.6 ± 1.0 mm (ABB). At baseline, the thickness of the buccal hard tissue at the level of the implant shoulder measured 1.1 ± 1.1 mm (BMP) and 1.4 ± 1.0 mm (ABB). At 3 years, it measured 0.9 ± 0.9 mm (BMP) and 0.7 ± 0.6 mm (ABB). CONCLUSIONS The present study demonstrated excellent implant survival rates and stable marginal hard tissue levels in both augmentation groups, 3 years after crown insertion. In addition, the clinical stability of soft and hard tissues was demonstrated in both groups

Bioinspired Diversification Approach Toward the Total Synthesis of Lycodine-Type Alkaloids.

Nitrogen heterocycles (azacycles) are common structural motifs in numerous pharmaceuticals, agrochemicals, and natural products. Many powerful methods have been developed and continue to be advanced for the selective installation and modification of nitrogen heterocycles through C-H functionalization and C-C cleavage approaches, revealing new strategies for the synthesis of targets containing these structural entities. Here, we report the first total syntheses of the lycodine-type Lycopodium alkaloids casuarinine H, lycoplatyrine B, lycoplatyrine A, and lycopladine F as well as the total synthesis of 8,15-dihydrohuperzine A through bioinspired late-stage diversification of a readily accessible common precursor, N-desmethyl-β-obscurine. Key steps in the syntheses include oxidative C-C bond cleavage of a piperidine ring in the core structure of the obscurine intermediate and site-selective C-H borylation of a pyridine nucleus to enable cross-coupling reactions

A Rational Active-Site Redesign Converts a Decarboxylase into a CC Hydratase: “Tethered Acetate” Supports Enantioselective Hydration of 4‑Hydroxystyrenes

The promiscuous regio- and stereoselective hydration of 4-hydroxystyrenes catalyzed by ferulic acid decarboxylase from <i>Enterobacter</i> sp. (FDC_<i>Es</i>) depends on bicarbonate bound in the active site, which serves as a proton relay activating a water molecule for nucleophilic attack on a quinone methide electrophile. This “cofactor” is crucial for achieving improved conversions and high stereoselectivities for (<i>S</i>)-configured benzylic alcohol products. Similar effects were observed with simple aliphatic carboxylic acids as additives. A rational redesign of the active site by replacing the bicarbonate or acetate “cofactor” with a newly introduced side-chain carboxylate from an adjacent amino acid yielded mutants that efficiently acted as CC hydratases. A single-point mutation of valine 46 to glutamate or aspartate improved the hydration activity by 40% and boosted the stereoselectivity 39-fold in the absence of bicarbonate or acetate

Metal Ion Promiscuity and Structure of 2,3‐Dihydroxybenzoic Acid Decarboxylase of Aspergillus oryzae

Broad substrate tolerance and excellent regioselectivity, as well as independence from sensitive cofactors have established benzoic acid decarboxylases from microbial sources as efficient biocatalysts. Robustness under process conditions makes them particularly attractive for preparative‐scale applications. The divalent metal‐dependent enzymes are capable of catalyzing the reversible non‐oxidative (de)carboxylation of a variety of electron‐rich (hetero)aromatic substrates analogously to the chemical Kolbe‐Schmitt reaction. Elemental mass spectrometry supported by crystal structure elucidation and quantum chemical calculations verified the presence of a catalytically relevant Mg$^{2+}$ complexed in the active site of 2,3‐dihydroxybenoic acid decarboxylase from Aspergillus oryzae (2,3‐DHBD_Ao). This unique example with respect to the nature of the metal is in contrast to mechanistically related decarboxylases, which generally have Zn$^{2+}$ or Mn$^{2+}$ as the catalytically active metal

Randomized, controlled clinical two-centre study using xenogeneic block grafts loaded with recombinant human bone morphogenetic protein-2 or autogenous bone blocks for lateral ridge augmentation

OBJECTIVES To test whether or not the use of a xenogeneic block loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) results in different bone quantity and quality compared to an autogenous bone block. MATERIALS AND METHODS Twenty-four patients with insufficient bone volume for implant placement were randomly assigned to two treatment modalities: a xenogeneic bone block loaded with rhBMP-2 (test) and an autogenous bone block (control). The horizontal ridge width was evaluated prior to augmentation, after augmentation and at 4 months. Patient-reported outcome measures (PROMs) were assessed at suture removal and at 4 months. Biopsies were obtained at 4 months and histologically evaluated. Intergroup comparisons were tested by a two-sided Wilcoxon-Mann-Whitney test, intra-group comparisons were performed with Wilcoxon-signed rank test, and all categorical variables were tested with Chi-squared tests. RESULTS One autogenous bone block failed. This patient was replaced, and in all subsequently treated 24 patients, implant placement was possible 4 months later. The median ridge width increased from 4.0 mm (Q1 = 2.0; Q3 = 4.0) (test) and 2.0 mm (Q1 = 2.0; Q3 = 3.0) (control) to 7.0 mm (Q1 = 6.0; Q3 = 8.0) (test) and 7.0 mm (Q1 = 6.0; Q3 = 8.0) (control) at 4 months (intergroup p > .05). A higher morbidity was reported at the augmented site in the control group during surgery. Sensitivity was more favourable in the test than that in the control group at 4 months. The biopsies revealed more mineralized tissue in the control group (p < .0043). CONCLUSIONS Both treatment modalities were successful in regenerating bone to place dental implants. PROMs did not reveal any significant differences between the groups except for pain during surgery at the recipient site (in favour of the test group). Histologically, a higher amount of mineralized tissue was observed for the control group at 4 months

Regioselective para-Carboxylation of Catechols by a Prenylated Flavin Dependent Decarboxylase

The utilization of CO$-2$ as a carbon source for organic synthesis meets the urgent demand for more sustainability in the production of chemicals. Herein, we report on the enzyme-catalyzed para-carboxylation of catechols, employing 3,4-dihydroxybenzoic acid decarboxylases (AroY) that belong to the UbiD enzyme family. Crystal structures and accompanying solution data confirmed that AroY utilizes the recently discovered prenylated FMN (prFMN) cofactor, and requires oxidative maturation to form the catalytically competent prFMN4$^{iminium}$ species. This study reports on the in vitro reconstitution and activation of a prFMN-dependent enzyme that is capable of directly carboxylating aromatic catechol substrates under ambient conditions. A reaction mechanism for the reversible decarboxylation involving an intermediate with a single covalent bond between a quinoid adduct and cofactor is proposed, which is distinct from the mechanism of prFMN-associated 1,3-dipolar cycloadditions in related enzymes