A genomic library from Lactobacillus delbrueckii subsp. bulgaricus was used to complement an Escherichia coli mutant strain deficient for both lactate dehydrogenase and pyruvate formate lyase, and thus unable to grow anaerobically. One recombinant clone was found to display a broad specificity NAD+-dependent D-2-hydroxyacid dehydrogenase activity. The corresponding gene (named hdhD) was subcloned and sequenced. The deduced amino acid sequence of the encoded enzyme indicates a 333-residue protein closely related to D-2-hydroxyisocaproate (i.e. 2-hydroxy-4-methyl-pentanoate) dehydrogenase (D-HO-HxoDH) of Lactobacillus casei and other NAD+-dependent D-lactate dehydrogenases (D-LDH) from several other bacterial species. The hdhD gene was overexpressed under the control of the lambda phage P, promoter and the enzyme was purified with a two-step method. The L. delbrueckii subsp. bulgaricus enzyme, like that of L. casei, was shown to be active on a wide variety of 2-oxoacid substrates except those having a branched P-carbon.NAD+-dependent 2-hydroxyacid dehydrogenases of broad substrate specificity have been reported in many species [l]. They catalyse the stereospecific and reversible reduction of various aliphatic and aromatic 2-oxocarboxylic acids to the corresponding 2-hydroxycarboxylic acids. The optically active products are not only valuable compounds as synthons for industrial processes as, for instance, in the production of semisynthetic antibiotics or pharmaceuticals [2], but they can also be used for clinical analysis of blood samples [3]. These enzymes can be divided into two groups on the basis of their substrate preference: mandelate dehydrogenase (or benzoylformate reductase), on the one hand, and 2-hydroxyisocaproate (i.e. 2-hydroxy-4-methylpentanoate) dehydrogenase (or 2-oxoisocaproate reductase), on the other hand. Each class can be further subdivided according to the D-or L-stereoisomer produced. Their actual substrate and function in vivo are not known.Lactobacillus confusus NAD'-dependent L-2-hydroxyisocaproate dehydrogenase (L-HO-HxoDH) was described for the first time in 1984 [4] and the corresponding gene was
Domain swapping in Ppl is determined by the balance of two opposing components of the free energy. One is the strain in the second beta turn that favors the dimer, and the other is the entropic cost of dimer formation that favors the monomer. A single-site mutation can disrupt this balance and trigger domain swapping.
Methyl 2-chloro-2-cyclopropylideneacetate (2) and its spiropentane analogue 3 cycloadd to dihydroisoquinoline N-oxide (9), pyrroline N-oxide (12), and C-phenyl-N-methylnitrone (16) to give 5-spirocyclopropaneisoxazolidines in good yields (58-93%). The thermal behavior of the 5-spirocyclopropaneisoxazolidines is rather differentiated, depending strongly on the constitution of the nitrone and the solvent. As nitrone 9 has the tendency to undergo cycloreversion reactions, the ketoamide rearrangement products 20 and 21 from its cycloadduct derive from the thermodynamically favored 4-spirocyclopropaneisoxazolidine regioisomers formed after the cycloreversion process. In DMSO as solvent different rearrangement processes take place, leading to benzoindolizinones in modest yields (15-21%). The cycloadducts from 12 and 16 undergo a cyclopropyl to cyclobutyl ring enlargement facilitated by the presence of a chlorine substituent on the carbon alpha to the spirocyclopropane ring. Whereas these compounds from nitrone 16 demonstrated an unusual stability, those from nitrone 12 undergo a cascade rearrangement to yield indolizinone derivatives 34, 35 cleanly (73-83% yield). This overall transformation offers a new method for the synthesis of the indolizine skeleton.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.