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Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging.

ABSTRACT: Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K- and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH. Both single amino acid replacements, M304R- and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.

SUBMITTER: Bogin O

PROVIDER: S-EPMC2373725 | biostudies-literature | 2002 Nov

REPOSITORIES: biostudies-literature

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Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging.

Bogin Oren O Levin Inna I Hacham Yael Y Tel-Or Shoshana S Peretz Moshe M Frolow Felix F Burstein Yigal Y

Protein science : a publication of the Protein Society 20021101 11

Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability ...[more]

PMID: 12381840

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Project description:The coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH) catalyzes a key reaction in the acetone- and butanol (solvent)-producing clostridia. It reduces acetyl-CoA and butyryl-CoA to the corresponding aldehydes, which are then reduced by alcohol dehydrogenase (ADH) to form ethanol and 1-butanol. The ALDH of Clostridium beijerinckii NRRL B593 was purified. It had no ADH activity, was NAD(H) specific, and was more active with butyraldehyde than with acetaldehyde. The N-terminal amino acid sequence of the purified ALDH was determined. The open reading frame preceding the ctfA gene (encoding a subunit of the solvent-forming CoA transferase) of C. beijerinckii NRRL B593 was identified as the structural gene (ald) for the ALDH. The ald gene encodes a polypeptide of 468 amino acid residues with a calculated M(r) of 51, 353. The position of the ald gene in C. beijerinckii NRRL B593 corresponded to that of the aad/adhE gene (encoding an aldehyde-alcohol dehydrogenase) of Clostridium acetobutylicum ATCC 824 and DSM 792. In Southern analyses, a probe derived from the C. acetobutylicum aad/adhE gene did not hybridize to restriction fragments of the genomic DNAs of C. beijerinckii and two other species of solvent-producing clostridia. In contrast, a probe derived from the C. beijerinckii ald gene hybridized to restriction fragments of the genomic DNA of three solvent-producing species but not to those of C. acetobutylicum, indicating a key difference among the solvent-producing clostridia. The amino acid sequence of the ALDH of C. beijerinckii NRRL B593 was most similar (41% identity) to those of the eutE gene products (CoA-acylating ALDHs) of Salmonella typhimurium and Escherichia coli, whereas it was about 26% identical to the ALDH domain of the aldehyde-alcohol dehydrogenases of C. acetobutylicum, E. coli, Lactococcus lactis, and amitochondriate protozoa. The predicted secondary structure of the C. beijerinckii ALDH suggests the presence of an atypical Rossmann fold for NAD(+) binding. A comparison of the proposed catalytic pockets of the CoA-dependent and CoA-independent ALDHs identified 6 amino acids that may contribute to interaction with CoA.

Project description:BackgroundCassava pulp is a promising starch-based biomasses, which consists of residual starch granules entrapped in plant cell wall containing non-starch polysaccharides, cellulose and hemicellulose. Strain CT4T, a novel mesophilic anaerobic bacterium isolated from soil collected from a cassava pulp landfill, has a strong ability to degrade polysaccharides in cassava pulp. This study explored a rarely described species within the genus Clostridium that possessed a group of cassava pulp-degrading enzymes.MethodsA novel mesophilic anaerobic bacterium, the strain CT4T, was identified based on phylogenetic, genomic, phenotypic and chemotaxonomic analysis. The complete genome of the strain CT4T was obtained following whole-genome sequencing, assembly and annotation using both Illumina and Oxford Nanopore Technology (ONT) platforms.ResultsAnalysis based on the 16S rRNA gene sequence indicated that strain CT4T is a species of genus Clostridium. Analysis of the whole-genome average amino acid identity (AAI) of strain CT4T and the other 665 closely related species of the genus Clostridium revealed a separated strain CT4T from the others. The results revealed that the genome consisted of a 6.3 Mb circular chromosome with 5,664 protein-coding sequences. Genome analysis result of strain CT4T revealed that it contained a set of genes encoding amylolytic-, hemicellulolytic-, cellulolytic- and pectinolytic enzymes. A comparative genomic analysis of strain CT4T with closely related species with available genomic information, C. amylolyticum SW408T, showed that strain CT4T contained more genes encoding cassava pulp-degrading enzymes, which comprised a complex mixture of amylolytic-, hemicellulolytic-, cellulolytic- and pectinolytic enzymes. This work presents the potential for saccharification of strain CT4T in the utilization of cassava pulp. Based on phylogenetic, genomic, phenotypic and chemotaxonomic data, we propose a novel species for which the name Clostridium manihotivorum sp. nov. is suggested, with the type strain CT4T (= TBRC 11758T = NBRC 114534T).

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Dataset Information

Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging.

Publications

Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging.

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