Hup-Type Hydrogenases of Purple Bacteria: Homology Modeling and Computational Assessment of Biotechnological Potential

Three-dimensional structures of six closely related hydrogenases from purple bacteria were modeled by combining the template-based and ab initio modeling approach. The results led to the conclusion that there should be a 4Fe3S cluster in the structure of these enzymes. Thus, these hydrogenases could draw interest for exploring their oxygen tolerance and practical applicability in hydrogen fuel cells. Analysis of the 4Fe3S cluster’s microenvironment showed intragroup heterogeneity. A possible function of the C-terminal part of the small subunit in membrane binding is discussed. Comparison of the built models with existing hydrogenases of the same subgroup (membrane-bound oxygen-tolerant hydrogenases) was carried out. Analysis of intramolecular interactions in the large subunits showed statistically reliable differences in the number of hydrophobic interactions and ionic interactions. Molecular tunnels were mapped in the models and compared with structures from the PDB. Protein–protein docking showed that these enzymes could exchange electrons in an oligomeric state, which is important for oxygen-tolerant hydrogenases. Molecular docking with model electrode compounds showed mostly the same results as with hydrogenases from E. coli, H. marinus, R. eutropha, and S. enterica; some interesting results were shown in case of HupSL from Rba. sphaeroides and Rvi. gelatinosus.

Hup-type Hydrogenases of Purple Bacteria: Homology Modeling and Computational Assessment of Biotechnological Potential

Three-dimensional structure of six closely related hydrogenases from purple bacteria has been modeled by combining template-based and ab initio modeling approach. The results lead to conclusion that there should be 4Fe3S-cluster in the structure of these enzymes. Thus, these hydrogenases could drive attention for exploring their oxygen tolerance and practical applicability in hydrogen fuel cells. Analysis of 4Fe3S-cluster's microenvironment showed intragroup heterogeneity. Possible function of the C-terminal part of the small subunit in membrane binding has been discussed. Comparison of the built models with existing hydrogenases of the same subgroup (membrane-bound oxygen-tolerant hydrogenases) has been carried out. Analysis of intramolecular interactions in the large subunits showed statistically reliable differences in number of hydrophobic interactions and number of ionic interactions. Molecular tunnels were mapped in the models and compared with structures from PDB. Protein-protein docking showed that these enzymes could exchange electrons in oligomeric state, which is important for oxygen-tolerant hydrogenases. Molecular docking with model electrode compounds showed mostly the same results as with hydrogenases from E.coli, H. marinus, R. eutropha, S. enterica; some interesting results were shown in case of HupSL from Rba. sphaeroides and Rvx. gelatinosus.

Molecular Tunnels in Enzymes and Thermophily: A Case Study on the Relationship to Growth Temperature

Developments in protein expression, analysis and computational capabilities are decisively contributing to a better understanding of the structure of proteins and their relationship to function. Proteins are known to be adapted to the growth rate of microorganisms and some microorganisms (named (hyper)thermophiles) thrive optimally at high temperatures, even above 100 °C. Nevertheless, some biomolecules show great instability at high temperatures and some of them are universal and required substrates and cofactors in multiple enzymatic reactions for all (both mesophiles and thermophiles) living cells. Only a few possibilities have been pointed out to explain the mechanisms that thermophiles use to successfully thrive under high temperatures. As one of these alternatives, the role of molecular tunnels or channels in enzymes has been suggested but remains to be elucidated. This study presents an analysis of channels in proteins (i.e., substrate tunnels), comparing two different protein types, glutamate dehydrogenase and glutamine phosphoribosylpyrophosphate amidotransferase, which are supposed to present a different strategy on the requirement for substrate tunnels with low and high needs for tunneling, respectively. The search and comparison of molecular tunnels in these proteins from microorganisms thriving optimally from 15 °C to 100 °C suggested that those tunnels in (hyper)thermophiles are required and optimized to specific dimensions at high temperatures for the enzyme glutamine phosphoribosylpyrophosphate amidotransferase. For the enzyme glutamate dehydrogenase, a reduction of empty spaces within the protein could explain the optimization at increasing temperatures. This analysis provides further evidence on molecular channeling as a feasible mechanism in hyperthermophiles with multiple relevant consequences contributing to better understand how they live under those extreme conditions.

Computational Investigation of O2 Diffusion Through an Intra–molecular Tunnel in AlkB; Influence of Polarization on O2 Transport

E. Coli AlkB catalyzes the direct dealkylation of various alkylated bases in damaged DNA. The diffusion of molecular Oxygen to the active site in AlkB is an essential step for the oxidative dealkylation activity. Despite detailed studies on the stepwise oxidation mechanism of AlkB, there is no conclusive picture of how O2 molecules reach the active site of the protein. Yu et al. (Nature,439, 879) proposed the existence of an intra–molecular tunnel based on their initial crystal structures of AlkB. We have employed computational simulations to investigate possible migration pathways inside AlkB for O2 molecules. Extensive molecular dynamics (MD) simulations, including explicit ligand sampling and potential of mean force (PMF) calculations, have been performed to provide a microscopic description of the O2 delivery pathway in AlkB. Analysis of intra–molecular tunnels using the CAVER software indicates two possible pathways for O2 to diffuse into the AlkB active site. Explicit ligand sampling simulations suggests that only one of these tunnels provides a viable route. The free energy path for an oxygen molecule to travel along each of these tunnels has been determined with AMBER and AMOEBA. Both PMFs indicate passive transport of O2 from the surface of the protein. However, the inclusion of explicit polarization shows a very large barrier for diffusion of the co–substrate out of the active site, compared with the non–polarizable potential. In addition, our results suggest that the mutation of a conserved residue along the tunnel, Y178, has dramatic effects on the dynamics of AlkB and on the transport of O2 along the tunnel.

Subunit Interactions and Glutamine Utilization byEscherichia coli Imidazole Glycerol Phosphate Synthase

ABSTRACT A selection strategy has been developed to identify amino acid residues involved in subunit interactions that coordinate the two half-reactions catalyzed by glutamine amidotransferases. The protein structures known for this class of enzymes have revealed that ammonia is shuttled over long distances and that each amidotransferase evolved different molecular tunnels for this purpose. The heterodimericEscherichia coli imidazole glycerol phosphate (IGP) synthase was probed to assess if residues in the substrate amination subunit (HisF) are critical for the glutaminase activity in the HisH subunit. The activity of the HisH subunit is dependent upon binding of the nucleotide substrate at the HisF active site. This regulatory function has been exploited as a biochemical selection of mutant HisF subunits that retain full activity with ammonia as a substrate but, when constituted as a holoenzyme with wild-type HisH, impair the glutamine-dependent activity of IGP synthase. The steady-state kinetic constants for these IGP synthases with HisF alleles showed three distinct effects depending upon the site of mutation. For example, mutation of the R5 residue has similar effects on the glutamine-dependent amidotransfer reaction; however,k cat/Km for the glutaminase half-reaction was increased 10-fold over that for the wild-type enzyme with nucleotide substrate. This site appears essential for coupling of the glutamine hydrolysis and ammonia transfer steps and is the first example of a site remote to the catalytic triad that modulates the process. The results are discussed in the context of recent X-ray crystal structures of glutamine amidotransferases that relate the glutamine binding and acceptor binding sites.