Dissertation/Thesis Abstract

Funktion, Modifikation und Anwendung von O2-toleranten, NAD(P)+-reduzierenden [NiFe]-Hydrogenasen
by Preissler, Janina, Dr.Nat., Technische Universitaet Berlin (Germany), 2018, 161; 11014031
Abstract (Summary)

Soluble, NAD(P)-dependent hydrogenases (SHs) catalyse the reversible splitting of H2 into two electrons and two protons followed by the reduction of the pyridine nucleotide cofactor. The two reactions take place in two different enzyme modules. The hydrogenase module consists of the subunits HoxHY and contains the active [NiFe]-center. The NAD+ reductase module comprised of HoxFU harbours a flavin close to the active site. A chain of [FeS] clusters electronically links both catalytic centers. Apart from the aerobic H2 oxidizer Ralstonia eutropha H16, there are further Knallgasbacteria such as Rhodococcus opacus and Hydrogenophilus thermoluteolus that possess SHs with extraordinary O2 tolerance, a very appealing feature for biotechnological applications. It has been shown that the SH from R. eutropha is well applicable for H2-driven cofactor recycling. While highly specific towards NAD+, its reactivity with NADP+ is very low. As a part of this work, the substrate spectrum of the ReSH was extended to NADP+ by rational mutagenesis resulting in a 240-fold higher catalytic efficiency for the new substrate. The SH variants with synthetic NADP+-reducing activity were characterized biochemically and electrochemically, and their functionality was challenged in coupled enzyme reactions. Apart from substrate specificity, improved thermostability and, e.g., salt tolerance is crucial for biotechnological application of SH. The thermophilic organism Hydrogenophilus thermoluteolus contains an SH (HtSH) with remarkable activity and stability at high temperatures. The HtSH was heterologously produced in R. eutropha, which enabled the first comprehensive enzymatic and spectroscopic study of this particular hydrogenase. In contrast to ReSH, the HtSH showed highest H2-dependent NAD+-reduction activity at 80°C and at pH 6.5. Infrared spectroscopic measurements revealed unusual vibrational stretching frequencies for the typical CO and CN- ligands of the H2-activating catalytic center, which differ from those known for ReSH. This indicates partially different structures/redox states of the active site. In case of the SHs from H. thermoluteolus and Rhodococcus opacus the H2-driven NAD+-reducing activity was found to depend strongly on the presence of divalent cations such as Ni2+ and Mg2+ in the enzyme assay. To obtain insight into this cation dependence, SH chimeras with mixed compositions of NAD+ reductase (HoxFU) and hydrogenase (HoxHY) modules of R. eutropha, R. opacus, and H. thermoluteolus were constructed. It turned out that the variants containing hydrogenase modules from either H. thermoluteolus or R. opacus in combination with the NAD+ reductase from R. eutropha showed cation-dependent, H2-driven NAD+ reduction activities. SH, chimeras carrying the hydrogenase module from R. eutropha were fully active also without additional Ni2+ and Mg2+. This identified the hydrogenase module to be responsible for the cation dependence. With exception of the SH-variant, which contains the hydrogenase module from H.thermoluteolus all chimeric SHs were able to facilitate autotrophic growth in R. eutropha under an atmosphere of H2, O2 and CO2. This subunit mixing system allows the optimization of biochemical features of the SH according to the desired reaction conditions. For example, the combination of highly NAD+-affine RoNAD+ reductase with the more active Rehydrogenase results in a chimeric SH that is not dependent on divalent cations in contrast to RoSH.

Indexing (document details)
Advisor: Lenz, Oliver
Commitee:
School: Technische Universitaet Berlin (Germany)
School Location: Germany
Source: DAI-C 81/1(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Bioengineering, Biochemistry
Keywords: Hydrogenases
Publication Number: 11014031
ISBN: 9781687915733
Copyright © 2019 ProQuest LLC. All rights reserved. Terms and Conditions Privacy Policy Cookie Policy
ProQuest