Biotin-dependent multifunctional enzymes carry out metabolically important carboxyl group transfer reactions and are potential targets for the treatment of obesity and type 2 diabetes. These enzymes use a tethered biotin cofactor to carry an activated carboxyl group between distantly spaced active sites. The mechanism of this transfer has remained poorly understood. Here we report the complete structure of pyruvate carboxylase at 2.0 angstroms resolution, which shows its domain arrangement. The structure, when combined with mutagenic analysis, shows that intermediate transfer occurs between active sites on separate polypeptide chains. In addition, domain rearrangements associated with activator binding decrease the distance between active-site pairs, providing a mechanism for allosteric activation. This description provides insight into the function of biotin-dependent enzymes and presents a new paradigm for multifunctional enzyme catalysis.

Previous studies showed that a series of purified condensed tannins (CTs) from warm-season perennial legumes exhibited high variability in their modulation of methane production during in vitro rumen digestion. The molecular weight differences between these CTs did not provide correlation with either the in vitro CH₄ production or the ability to precipitate bovine serum albumin. In an effort to delineate other structure-activity relationships from these methane abatement experiments, the structures of purified CTs from these legumes were assessed with a combination of methanolysis, quantitative thiolysis, ¹H-¹³C HSQC NMR spectroscopy and ultrahigh-resolution MALDI-TOF MS. The composition of these CTs is very diverse: procyanidin/prodelphinidin (PC/PD) ratios ranged from 98/2 to 2/98; cis/trans ratios ranged from 98/2 to 34/66; mean degrees of polymerization ranged from 6 to 39; and % galloylation ranged from 0 to 75%. No strong correlation was observed between methane production and the protein precipitation capabilities of the CT towards three different proteins (BSA, lysozyme, and alfalfa leaf protein) at ruminal pH. However, a strong non-linear correlation was observed for the inhibition of methane production versus the antioxidant activity in plant sample containing typical PC- and PD-type CTs. The modulation of methane production could not be correlated to the CT structure (PC/PD or cis/trans ratios and extent of galloylation). The most active plant in methane abatement was Acacia angustissima, which contained CT, presenting an unusual challenge as it was resistant to standard thiolytic degradation conditions and exhibited an atypical set of cross-peak signals in the 2D NMR. The MALDI analysis supported a 5-deoxy flavan-3-ol-based structure for the CT from this plant.

The nature of hydrogen bonding within intermolecular complexes of carboxylic acids and 1-methylimidazole (1-MeIm), 1-n-butylimidazole (1-BuIm), and 1-tert-butylimidazole (1-t-BuIm) in chloroform was characterized by Fourier transform infrared spectroscopy. Earlier spectroscopic studies indicated that carboxylic acid–1-MeIm complexes are of three types: (I) neutral complexes with the weaker acids (pK_a 2.2) in which the antisymmetric carbonyl stretching frequencies are lowered relative to the free acids and the ethyl esters of the acids; (II) ionic complexes of stronger acids (pK_a ⩽ 2.0) in which the carbonyl stretching frequencies are slightly lower than those for the tetrabutylammonium salts of the acids; (III) depolarized partially ionic complexes coexisting with type II, in which the carbonyl stretching frequencies are intermediate between those for the tetrabutylammonium salts (bond order 1.5) and the free acids (bond order 2.0).¹² Assignment of the ionic and intermediate carbonyl stretching frequencies was verified by shifts to longer wavelengths in the ¹⁸O-labeled 2,2-dichloropropanoic acid–1-MeIm complexes. Type III complexes have been postulated to incorporate a low barrier hydrogen bond (LBHB) between the N³ of the imidazole ring and the carboxylic group. The size of the alkyl group in 1-alkylimidazoles has no significant effect on the carbonyl stretching frequencies in any of the complexes. However, increasing bulkiness in the alkyl group increases the intensity of the type III species relative to type II, so that the equilibrium is shifted toward low barrier hydrogen bonding in solution. The broad bands at approximately 2500 and 1900 cm^–1 in the R-COOH–1-alkylimidazole complexes are similar to those classically attributed to strong hydrogen bonds. These bands are absent from the spectra of R-COOD–1-alkylimidazole complexes. Moreover, the antisymmetric carbonyl stretching bands characteristic of the type III, or LBHB-bonded complexes, are greatly decreased in intensity in the spectra of R-COOD–1-alkylimidazole complexes, and are shifted to higher wavelengths nearer to those expected for the free R-COOD. The deuteron is more strongly attached to oxygen, whereas the corresponding proton is more free to engage in low-barrier hydrogen bonding. Spectroscopic data indicate that R-COOH–1-alkylimidazole complexes are unexpectedly strong in CHCl₃, perhaps because of resonance assisted hydrogen bonding.