Suchergebnisse

D-amino acid oxidase (DAAO) plays an important role in the functioning of
both prokaryotes and eukaryotes. DAAO is increasingly being used in practice, including
for the determination of D-amino acids in complex samples, including human tissues
and fl uids. There are generally two types of DAAO in all organisms. The fi rst type is
an enzyme highly specifi c for D-aspartate and has its own name D-aspartate oxidase
(DASPO). DAAO of the second type is characterized by a wide spectrum of substrate
specificity, with preference for one or another D-amino acid varying from source to
source. The activity of DAAO with a large number of substrates greatly complicates the
selective determination of a particular D-amino acid. The problem is often solved by
choosing an enzyme that, under the conditions of analysis, has low or no activity with
other D-amino acids present in the sample. For the convenience of selecting a particular
enzyme, we have collected and analyzed literature data on the catalytic parameters
of known DAAOs with the most important D-amino acids. In addition, similar data
are presented for novel recombinant DAAOs from the methylotrophic yeast Ogataea
parapolymorpha DL-1. Analysis of the data shows that, with the D-amino acid series,
the new OpaDASPO and OpaDAAO have the highest catalytic parameters.

NAD(P)+
-dependent formate dehydrogenase (FDH, EC 1.2.1.2.) catalyzes the oxidation of formate ion with the coupled reduction of NAD(P)+
to
NAD(P)H. Previously, in our laboratory, a genetic construct was obtained with
the soyfdh2 gene encoding isoenzyme 2 of formate dehydrogenase from soybean
Glycine max (SoyFDH). In this construct the nucleotide sequence encoding the
signal peptide responsible for the transport of the pro-enzyme into the mitochondria of plant cells (the SoyFDH_L enzyme) was deleted. In this work, a second variant of SoyFDH_S was obtained, in which, compared to SoyFDH_L, the sequence
at the N-terminus was reduced and changed to mimic the N-terminus sequence in
FDH from Pseudomonas sp.101 bacteria. Next, a sequence of six histidine residues
(His-tag) was added to the N-terminus of the long and short forms of SoyFDH.
All four SoyFDH variants were expressed in E. coli BL21(DE3)CodonPlus, these
enzymes were purified, their kinetic parameters were determined, and thermal
stability was studied. In the case of SoyFDH_L, which is similar to the natural
variant of the enzyme, both with and without His-tag, the expression level is two
times higher compared to the truncated variant. The addition of His-tag to the
N-terminus of enzymes reduces the level of expression. Changing the sequence
of the N-terminus, as well as introducing the His-tag sequence to the N-terminus,
does not significantly affect thermal stability of the enzymes at temperatures of
50–56 °C. However, due to the higher values of the activation enthalpy ΔH≠
of the
thermal inactivation process, the shortened form at normal temperatures is 3 times
more stable than the natural one. A comparison of the kinetic parameters of the two SoyFDH variants shows that the catalytic constants are the same, but the long
version of SoyFDH_L has lower values KM
HCOO–
, and the short version has lower
KM
NAD+
values. The introduction of His-tag into the N-terminus of enzymes does not
affect their kinetic parameters.

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from methylotrophic bacterium Pseudomonas sp.101 (PseFDH) has one of the highest thermal stability among all known enzymes of this group. The introduction of a number of amino acid substitutions into PseFDH made it possible to obtain a multipoint mutant PseFDH SM4S enzyme with even higher temperature and chemical stability. Previously, we showed that the introduction of additional single point replacements S131A, or S160A, or E170D into PseFDH SM4S led to further stabilization of the enzyme. In this work, based on the PseFDH SM4S S131A mutant, new mutant FDHs obtained, in which, compared to PseFDH SM4S, we added double S131A/E170D (M2), triple S131A/S160A/E170D (M3) and quadruple S131A/S160A/ E170D/S145A (PseFDH SM4A M3) amino acids replacements. The new PseFDH mutants were overexpressed in E. coli cells, puri ed and characterized. The S131A/E170D and S131A/S160A/E170D changes provided further improving thermal stability. The introduction of the S145A substitution into PseFDH SM4S M4 leads to a signi cant decrease in KMNAD+ and KMHCOO- while maintaining the catalytic constant at the same level. This mutant form can be successfully used in NADH regeneration systems, as well as for the detection of NAD+ and formate in biological systems.

Our earlier annotation of genome of the yeast Ogataea parapolymorpha DL-1 made it possible to identify ve genes of potential D-amino acids oxidases. All opadaao1 - opadaao5 genes were cloned and expressed in E. coli. Four OpaDAAO1- OpaDAAO4 enzymes were obtained in highly puri ed form and their catalytic properties were studied. It was found that among all DAAOs described in the literature so far, the enzyme OpaDAAO1 has the highest catalytic constant kcat with D-Ala, which makes it promising for practical applications. However, in addition to good catalytic parameters, effective application of the enzyme in practice requires high stability and knowledge of the inactivation mechanism, including at elevated temperatures. In this work we studied the effect of elevated temperatures on the stability of OpaDAAO1. The enzyme was shown to have high thermal stability in comparison with majority of other D-amino acid oxidases. The kinetics of OpaDAAO1 inactivation at different temperatures, initial concentrations of the enzyme, and in the presence of exogenous FAD were studied. A possible kinetic scheme of inactivation was proposed based on the data obtained.