Crystal structures and kinetics of Type III 3-phosphoglycerate dehydrogenase reveal catalysis by lysine.

D-Phosphoglycerate dehydrogenase (PGDH) catalyzes the first committed step of the phosphorylated serine biosynthesis pathway. Here, we report for the first time, the crystal structures of Type IIIK PGDH from Entamoeba histolytica in the apo form, as well as in complexes with substrate (3-phosphoglyceric acid) and cofactor (NAD(+) ) to 2.45, 1.8 and 2.2 Å resolution, respectively. Comparison of the apo structure with the substrate-bound structure shows that the substrate-binding domain is rotated by ~ 20° to close the active-site cleft. The cofactor-bound structure also shows a closed-cleft conformation, in which NAD(+) is bound to the nucleotide-binding domain and a formate ion occupies the substrate-binding site. Superposition of the substrate- and cofactor-bound structures represents a snapshot of the enzyme in the active form, where C2 of the substrate and C4N of the cofactor are 2.2 Å apart, and the amino group of Lys263 is close enough to the substrate to remove the proton from the hydroxyl group of PGA, indicating the role of Lys in the catalysis. Mutation of Lys263 to Ala yields just 0.8% of the specific activity of the wild-type enzyme, revealing that Lys263 indeed plays an integral role in the catalytic activity. The detectable activity of the mutant, however, indicates that after 20° rotation of the substrate-binding domain, the resulting positions of the substrate and cofactor are sufficiently close to make a productive reaction.

Characterization of carbonic anhydrase from diversified genus for biomimetic carbon-dioxide sequestration.

Diversified group of bacteria were screened for carbonic anhydrase (CA) activity. Significant CA activity was found in crude enzyme extracts of Enterobacter and Aeromonas isolates while minimal or negligible CA activity was observed in case of Shigella and Klebsiella spp. Optimization and characterization study of potent CA producing isolates revealed that the maximum enzyme activity of 3.86 EU/ml was observed in E. taylorae and the optimum pH range for enzyme stability was found to be 7.5-9.0 along with an optimum temperature range of 35-50 °C. The molecular mass of CA was 29-kDa indicating α-type with periplasmic and cytosolic location. Present investigation for the first time reports CA in diversified genus and optimized parameters for enhanced production of CA in Enterobacter sp. & Aeromonas sp. from fresh water bodies that inturn lay down grounds for exploitation of CA from E. taylorae as an efficient catalyst for CO(2) sequestration within a bioreactor.

Helicobacter pylori cagA, vacA and iceA genotypes in western Indian population of Maharashtra with varied gastroduodenal diseases.

Scarce reports relying on rapid urease test, serology and histopathology are currently known for H. pylori from Western India, Maharashtra. We investigated H. pylori genotypes at molecular level in gastro-duodenal disease population during the years 2002-2005. H. pylori presence was scored by polymerase chain reaction in the infected biopsies (n = 95) in various gastric diseases. H. pylori specific 16S rDNA gene amplification based preliminary identification coupled with protein coding gene amplification scores were assessed for the incidence. H. pylori 16S rDNA and 7 housekeeping genes were detected in all biopsies, whereas 71.18% and 28% found to be cagA positive and negative respectively. The vacA toxigenic alleles (vacA s1) and middle region subunit vac m1a were found in 54%, and 59% patients. However, the iceA1 was present in 40.06%; the iceA2 was less i.e. in 13.5% patients. The most common allelic combinations in different age groups irrespective of disease types were 13-30, 31-45, 46-60 and 61-73 were cagA-vac m1a-vacA s1-iceA1. In our analysis, PCR was found to be 100% accurate in detecting H. pylori in gastric biopsies. Among West Indian population H. pylori was found to be present, irrespective of any correlation with the genotype and gender of patients with the clinical outcome. However, the genotype incidences were related to age of the patients, wherein the age group ranging from 46 to 60 years was found be susceptible for H. pylori infection.