The Isoenzymic Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453—An Episodic History and Still Mysterious after 60 Years

Researching the involvement of molecular oxygen in the degradation of the naturally occurring bicyclic terpene camphor has generated a six-decade history of fascinating monooxygenase biochemistry. While an extensive bibliography exists reporting the many varied studies on camphor 5-monooxygenase, the initiating enzyme of the relevant catabolic pathway in Pseudomonas putida ATCC 17453, the equivalent recorded history of the isoenzymic diketocamphane monooxygenases, the enzymes that facilitate the initial ring cleavage of the bicyclic terpene, is both less extensive and more enigmatic. First referred to as ‘ketolactonase—an enzyme for cyclic lactonization’—the enzyme now classified as 2,5-diketocamphane 1,2-monooxygenase (EC 1.14.14.108) holds a special place in the history of oxygen-dependent biochemistry, being the first biocatalyst confirmed to undertake a biooxygenation reaction equivalent to the peracid-catalysed Baeyer–Villiger chemical oxidation first reported in the late 19th century. However, following that auspicious beginning, the biochemistry of EC 1.14.14.108, and its isoenzymic partner 3,6-diketocamphane 1,6-monooxygenase (EC 1.14.14.155) was dogged for many years by the mistaken belief that the enzymes were true flavoproteins that function with a tightly-bound flavin cofactor in the active site. This misconception led to a number of erroneous interpretations of relevant experimental data. It is only in the last decade, initially as the result of pure serendipity, that these enzymes have been confirmed to be members of a relatively recently discovered class of oxygen-dependent enzymes, the flavin-dependent two-component monooxygenases. This has promoted a renaissance of interest in the enzymes, resulting in programmes of research that have significantly expanded current knowledge of both their mode of action and regulation in camphor-grown P. putida ATCC 17453. However, some features of the biochemistry of the isoenzymic diketocamphane monooxygenases remain currently unexplained. It is the episodic history of these enzymes and some of what remains unresolved that are the principal subjects of this review.

Application of Oxidative Ring Opening/Ring Closing by Reductive Amination Protocol for the Stereocontrolled Synthesis of Functionalized Azaheterocycles

The current Account gives an insight into the synthesis of some N-heterocyclic β-amino acid derivatives and various functionalized saturated azaheterocycles accessed from substituted cycloalkenes via ring C=C bond oxidative cleavage followed by ring closing across double reductive amination. The ring-cleavage protocol has been accomplished according to two common approaches: a) Os-catalyzed dihydroxylation/NaIO4 vicinal diol oxidation and b) ozonolysis. A comparative study on these methodologies has been investigated. Due to the everincreasing relevance of organofluorine chemistry in drug research as well as of the high biological potential of β-amino acid derivatives several illustrative examples to the access of various fluorine-containing piperidine or azepane β-amino acid derivatives are also presented in the current Account.1 Introduction2 Olefin-Bond Transformation by Oxidative Ring Cleavage3 Synthesis of Saturated Azaheterocycles via Oxidative Ring-Opening/Ring-Closing Double Reductive Amination3.1 Importance of Fluorine-Containing Azaheterocycles in Pharmaceutical Research3.2 Synthesis of Azaheterocyclic Amino Acid Derivatives with a Piperidine or Azepane Framework through Oxidative Ring Opening/Reductive Amination3.2.1 Synthesis of Piperidine β-Amino Esters3.2.2 Synthesis of Azepane β-Amino Esters3.2.3 Synthesis of Fluorine-Containing Piperidine γ-Amino Esters3.3 Synthesis of Tetrahydroisoquinoline Derivatives through Oxidative Ring Opening/Reductive Amination Protocol3.4 Synthesis of Functionalized Benzazepines through Reductive Amination3.4.1 Synthesis of Benzo[c]azepines3.4.2 Synthesis of Benzo[d]azepines3.5 Synthesis of Various N-Heterocycles via Ozonolysis/Reductive Amination3.5.1 Synthesis of Compounds with an Azepane Ring3.5.2 Synthesis of Piperidine β-Amino Acids and Piperidine-Fused β-Lactams3.5.3 Synthesis of γ-Lactams with a Piperidine Ring3.5.4 Synthesis of other N-Heterocycles4 Summary and Outlook5 List of Abbreviations

Advances in engineering microbial biosynthesis of aromatic compounds and related compounds

Aromatic compounds have broad applications and have been the target of biosynthetic processes for several decades. New biomolecular engineering strategies have been applied to improve production of aromatic compounds in recent years, some of which are expected to set the stage for the next wave of innovations. Here, we will briefly complement existing reviews on microbial production of aromatic compounds by focusing on a few recent trends where considerable work has been performed in the last 5 years. The trends we highlight are pathway modularization and compartmentalization, microbial co-culturing, non-traditional host engineering, aromatic polymer feedstock utilization, engineered ring cleavage, aldehyde stabilization, and biosynthesis of non-standard amino acids. Throughout this review article, we will also touch on unmet opportunities that future research could address.

Domino Reactions of Chromones with Heterocyclic Enamines

Domino reactions of heterocyclic enamines with chromone derivatives provides a convenient synthesis of a great variety of annulated heterocyclic ring systems. The course of the reaction depends on the type of substituent located at position 3 of the chromone. Reactions of 3-unsubstituted chromones, 3-nitrochromones, and 3-halochromones proceed by conjugate addition of the carbon atom of the enamine to carbon C-2 of the chromone, ring cleavage, and recyclization via the chromone carbonyl group. In the case of 3-formylchromes, 3-dichloroacetylchromone, 3-perfluoroalkanoylthiochromones, 3-(2-fluorobenzoyl)chromones, and 3-methoxalylchromones the final cyclization proceeds via the carbonyl group located outside the chromone moiety. The functional groups located at the carbonyl group at position 3 of the chromone allow for further synthetic transformations including additional ring closures.Contents1 Introduction2 3-Unsubstituted Chromones3 3-Nitrochromones4 3-Formylchromes5 3-Dichloroacetylchromone6 3-Perfluoroalkanoylthiochromones7 3-Methoxalylchromones8 3-(2-Fluorobenzoyl)chromones9 3-Halochromones10 Chromone-3-carboxylic Acids11 Conclusions

Differential Roles of Three Different Upper Pathway Meta Ring Cleavage Product Hydrolases in the Degradation of Dibenzo- p -dioxin and Dibenzofuran by Sphingomonas wittichii RW1

Sphingomonas wittichii RW1 grows on the two related compounds dibenzofuran (DBF) and dibenzo- p -dioxin (DXN) as the sole source of carbon. Previous work by others (P.V. Bunz, R. Falchetto, and A.M. Cook. Biodegradation 4:171-8, 1993, doi: 10.1007/BF00695119) identified two upper pathway meta cleavage product hydrolases (DxnB1 and DxnB2) active on the DBF upper pathway metabolite 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)-hexa-2,4-dienoate. We took a physiological approach to determine the role of these two enzymes in the degradation of DBF and DXN by RW1. Single knockouts of either plasmid located dbfB1 or chromosome located dbfB2 had no effect on RW1 growth on either DBF or DXN. However, a double knockout lost the ability to grow on DBF but still grew normally on DXN demonstrating that DbfB1 and DbfB2 are the only hydrolases involved in the DBF upper pathway. Using a transcriptomic-guided approach we identified a constitutively expressed third hydrolase encoded by the chromosomally located SWIT0910 gene. Knockout of SWIT0910 resulted in a strain that no longer grows on DXN but still grows normally on DBF. Thus the DbfB1 and DbfB2 hydrolases function in the DBF but not the DXN catabolic pathway and the SWIT0190 hydrolase functions in the DXN but not the DBF catabolic pathway. Importance S. wittichii RW1 is one of only a few strains known to grow on DXN as the sole course of carbon. Much of the work deciphering the related RW1 DXN and DBF catabolic pathways has involved genome gazing, transcriptomics, proteomics, heterologous expression, and enzyme purification and characterization. Very little research has utilized physiological techniques to precisely dissect the genes and enzymes involved in DBF and DXN degradation. Previous work by others identified and extensively characterized two RW1 upper pathway hydrolases. Our present work demonstrates that these two enzymes are involved in DBF but not DXN degradation. In addition, our work identified a third constitutively expressed hydrolase that is involved in DXN but not DBF degradation. Combined with our previous work, this means that the RW1 DXN upper pathway involves genes from three very different locations in the genome: an initial plasmid-encoded dioxygenase and a ring cleavage enzyme and hydrolase encoded on opposite sides of the chromosome.