Gary W. Black

The Location of the Ligand-binding Site of Carbohydrate-binding Modules That Have Evolved from a Common Sequence Is Not Conserved

Polysaccharide-degrading enzymes are generally modular proteins that contain non-catalytic carbohydrate-binding modules (CBMs), which potentiate the activity of the catalytic module. CBMs have been grouped into sequence-based families, and three-dimensional structural data are available for half of these families. Clostridium thermocellum xylanase 11A is a modular enzyme that contains a CBM from family 6 (CBM6), for which no structural data are available. We have determined the crystal structure of this module to a resolution of 2.1 Å. The protein is a β-sandwich that contains two potential ligand-binding clefts designated cleft A and B. The CBM interacts primarily with xylan, and NMR spectroscopy coupled with site-directed mutagenesis identified cleft A, containing Trp-92, Tyr-34, and Asn-120, as the ligand-binding site. The overall fold of CBM6 is similar to proteins in CBM families 4 and 22, although surprisingly the ligand-binding site in CBM4 and CBM22 is equivalent to cleft B in CBM6. These structural data define a superfamily of CBMs, comprising CBM4, CBM6, and CBM22, and demonstrate that, although CBMs have evolved from a relatively small number of ancestors, the structural elements involved in ligand recognition have been assembled at different locations on the ancestral scaffold.

Convergent evolution sheds light on the anti-β-elimination mechanism common to family 1 and 10 polysaccharide lyases

Enzyme-catalyzed β-elimination of sugar uronic acids, exemplified by the degradation of plant cell wall pectins, plays an important role in a wide spectrum of biological processes ranging from the recycling of plant biomass through to pathogen virulence. The three-dimensional crystal structure of the catalytic module of a “family PL-10” polysaccharide lyase, Pel10Acm from Cellvibrio japonicus, solved at a resolution of 1.3 Å, reveals a new polysaccharide lyase fold and is the first example of a polygalacturonic acid lyase that does not exhibit the “parallel β-helix” topology. The “Michaelis” complex of an inactive mutant in association with the substrate trigalacturonate/Ca2+ reveals the catalytic machinery harnessed by this polygalacturonate lyase, which displays a stunning resemblance, presumably through convergent evolution, to the tetragalacturonic acid complex observed for a structurally unrelated polygalacturonate lyase from family PL-1. Common coordination of the −1 and +1 subsite saccharide carboxylate groups by a protein-liganded Ca2+ ion, the positioning of an arginine catalytic base in close proximity to the α-carbon hydrogen and numerous other conserved enzyme–substrate interactions, considered in light of mutagenesis data for both families, suggest a generic polysaccharide anti-β-elimination mechanism.

DNA sequence of the cut A, B and C genes, encoding the molybdenum containing hydroxylase carbon monoxide dehydrogenase, from Pseudomonas thermocarboxydovorans strain C2.

Pseudomonas thermocarboxydovorans strain C2 is capable of using carbon monoxide as the sole source of carbon and energy. The key enzyme for CO utilisation is the molybdenum containing iron-flavoprotein carbon monoxide dehydrogenase (CODH). This paper reports the DNA sequencing of a 4.7 kb region of the C2 genome which appears to encode the CODH enzyme. The genes for the three subunits of CODH, which we have named cut A, B and C, have been identified and they appear to form an operon. The predicted protein sequences of the three subunits have homology to the structurally related protein, xanthine dehydrogenase, from Drosophila melanogaster. By comparison with xanthine dehydrogenase it can be predicted that the molybdenum cofactor binds to the large subunit of CODH, the small subunit of CODH contains the iron-sulphur centers and the medium subunit binds FAD/NAD+.

Bioinformatic insights into the biosynthesis of the Group B carbohydrate in Streptococcus agalactiae.

Streptococcus agalactiae is a major human and animal pathogen, most notable as a cause of life-threatening disease in neonates. S. agalactiae is also called the Group B Streptococcus in reference to the diagnostically significant Lancefield Group B typing antigen. Although the structure of this complex carbohydrate antigen has been solved, little is known of its biosynthesis beyond the identification of a relevant locus in sequenced S. agalactiae genomes. Analysis of the sugar linkages present in the Group B carbohydrate (GBC) structure has allowed us to deduce the minimum enzymology required to complete its biosynthesis. Most of the enzymes required to complete this biosynthesis can be identified within the putative biosynthetic locus. Surprisingly, however, three crucial N-acetylglucosamine transferases and enzymes required for activated precursor synthesis are not apparently located in this locus. A model for GBC biosynthesis wherein the complete polymer is assembled at the cytoplasmic face of the plasma membrane before translocation to the cell surface is proposed. These analyses also suggest that GBC is the major teichoic acid-like polymer in the cell wall of S. agalactiae, whereas lipoteichoic acid is the dominant poly(glycerophosphate) antigen. Genomic analysis has allowed us to predict the pathway leading to the biosynthesis of GBC of S. agalactiae.

Probing the enantioselectivity of a diverse group of purified cobalt-centred nitrile hydratases

In this study a diverse range of purified cobalt containing nitrile hydratases (NHases, EC 4.2.1.84) from Rhodopseudomonas palustris HaA2 (HaA2), Rhodopseudomonas palustris CGA009 (009), Sinorhizobium meliloti 1021 (1021), and Nitriliruptor alkaliphilus (iso2), were screened for the first time for their enantioselectivity towards a broad range of chiral nitriles. Enantiomeric ratios of >100 were found for the NHases from HaA2 and CGA009 on 2-phenylpropionitrile. In contrast, the Fe-containing NHase from the well-characterized Rhodococcus erythropolis AJ270 (AJ270) was practically aselective with a range of different α-phenylacetonitriles. In general, at least one bulky group in close proximity to the α-position of the chiral nitriles seemed to be necessary for enantioselectivity with all NHases tested. Nitrile groups attached to a quaternary carbon atom were only reluctantly accepted and showed no selectivity. Enantiomeric ratios of 80 and >100 for AJ270 and iso2, respectively, were found for the pharmaceutical intermediate naproxennitrile, and 3-(1-cyanoethyl)benzoic acid was hydrated to the corresponding amide by iso2 with an enantiomeric ratio of >100.

Conformational effects on the circular dichroism of Human Carbonic Anhydrase II: a multilevel computational study.

Circular Dichroism (CD) spectroscopy is a powerful method for investigating conformational changes in proteins and therefore has numerous applications in structural and molecular biology. Here a computational investigation of the CD spectrum of the Human Carbonic Anhydrase II (HCAII), with main focus on the near-UV CD spectra of the wild-type enzyme and it seven tryptophan mutant forms, is presented and compared to experimental studies. Multilevel computational methods (Molecular Dynamics, Semiempirical Quantum Mechanics, Time-Dependent Density Functional Theory) were applied in order to gain insight into the mechanisms of interaction between the aromatic chromophores within the protein environment and understand how the conformational flexibility of the protein influences these mechanisms. The analysis suggests that combining CD semi empirical calculations, crystal structures and molecular dynamics (MD) could help in achieving a better agreement between the computed and experimental protein spectra and provide some unique insight into the dynamic nature of the mechanisms of chromophore interactions.

The impact of pH and nutrient stress on the growth and survival of Streptococcus agalactiae

Streptococcus agalactiae is a major neonatal pathogen that is able to colonise various host environments and is associated with both gastrointestinal and vaginal maternal carriage. Maternal vaginal carriage represents the major source for transmission of S. agalactiae to the foetus/neonate and thus is a significant risk factor for neonatal disease. In order to understand factors influencing maternal carriage we have investigated growth and long term survival of S. agalactiae under conditions of low pH and nutrient stress in vitro. Surprisingly, given that vaginal pH is normally <4.5, S. agalactiae was found to survive poorly at low pH and failed to grow at pH 4.3. However, biofilm growth, although also reduced at low pH, was shown to enhance survival of S. agalactiae. Proteomic analysis identified 26 proteins that were more abundant under nutrient stress conditions (extended stationary phase), including a RelE family protein, a universal stress protein family member and four proteins that belong to the Gls24 (PF03780) stress protein family. Cumulatively, these data indicate that novel mechanisms are likely to operate that allow S. agalactiae survival at low pH and under nutrient stress during maternal vaginal colonisation and/or that the bacteria may access a more favourable microenvironment at the vaginal mucosa. As current in vitro models for S. agalactiae growth appear unsatisfactory, novel methods need to be developed to study streptococcal colonisation under physiologically-relevant conditions.

A proteomic investigation of Streptococcus agalactiae reveals that human serum induces the C protein β antigen and arginine deiminase.

Streptococcus agalactiae is a major neonatal pathogen. Disease progression is characterised by bacterial adaptation from commensal maternal vaginal colonisation to environments associated with neonatal disease, including exposure to blood. To explore this adaptation in vitro, we have used proteomics to identify proteins differentially expressed following growth on Todd Hewitt agar in the presence or absence of 10% v/v human serum. Twelve differentially expressed proteins were identified. Notably, the C protein β antigen and arginine deiminase proteins were upregulated following growth in the presence of human serum, consistent with previous studies implicating these two proteins in the pathogenesis of S. agalactiae disease.

The effect of Maillard reaction products and yeast strain on the synthesis of key higher alcohols and esters in beer fermentations

The effect of Maillard reaction products (MRPs), formed during the production of dark malts, on the synthesis of higher alcohols and esters in beer fermentations was investigated by headspace solid-phase microextraction GC-MS. Higher alcohol levels were significantly (p<0.05) higher in dark malt fermentations, while the synthesis of esters was inhibited, due to possible suppression of enzyme activity and/or gene expression linked to ester synthesis. Yeast strain also affected flavour synthesis with Saccharomyces cerevisiae strain A01 producing considerably lower levels of higher alcohols and esters than S288c and L04. S288c produced approximately double the higher alcohol levels and around twenty times more esters compared to L04. Further investigations into malt type-yeast strain interactions in relation to flavour development are required to gain better understanding of flavour synthesis that could assist in the development of new products and reduce R&D costs for the industry.

Structural, functional, and mutagenesis studies of UDP-glycosyltransferases

The biosynthesis of the complex carbohydrates that govern many cellular functions requires the action of a diverse range of selective glycosyltransferases (GTs). Uridine diphosphate sugar-utilizing GTs (UGTs) account for the majority of characterized GTs. GTs have been classified into families (currently 92) based on amino-acid sequence similarity. However, as amino-acid sequence similarity cannot reliable predict catalytic mechanism, GTs have also been grouped into four clans based on catalytic mechanism and structural fold. GTs catalyze glycosidic bond formation with two possible stereochemical outcomes: inversion or retention of anomeric configuration. All UGTs also belong to one of two distinct structural folds, GT-A and GT-B. UGTs have conserved residues that are associated with nucleotide diphosphate sugar recognition and acceptor recognition. UGT diversification has been performed using in vitro DNA recombination, domain swapping, and random mutagenesis.