Marleen Otzen

Hansenula polymorpha Pex20p is an oligomer that binds the peroxisomal targeting signal 2 (PTS2)

We have cloned and characterized the Hansenula polymorpha PEX20 gene. The HpPEX20 gene encodes a protein of 309 amino acids (HpPex20p) with a calculated molecular mass of ∼35 kDa. In cells of an HpPEX20 disruption strain, PTS2 proteins were mislocalized to the cytosol, whereas PTS1 matrix protein import proceeded normally. Also, the PTS2 proteins amine oxidase and thiolase were normally assembled and active in these cells, suggesting HpPex20p is not involved in oligomerization/activation of these proteins. Localization studies revealed that HpPex20p is predominantly associated with peroxisomes. Using fluorescence correlation spectroscopy we determined the native molecular mass of purified HpPex20p and binding of a synthetic peptide containing a PTS2 sequence. The data revealed that purified HpPex20p forms oligomers, which specifically bind PTS2-containing peptides.

Peroxisome fission in Hansenula polymorpha requires Mdv1 and Fis1, two proteins also involved in mitochondrial fission

We show that Mdv1 and Caf4, two components of the mitochondrial fission machinery in Saccharomyces cerevisiae, also function in peroxisome proliferation. Deletion of MDV1, CAF4 or both, however, had only a minor effect on peroxisome numbers at peroxisome‐inducing growth conditions, most likely related to the fact that Vps1 – and not Dnm1 – is the key player in peroxisome fission in this organism. In contrast, in Hansenula polymorpha, which has only a Dnm1‐dependent peroxisome fission machinery, deletion of MDV1 led to a drastic reduction of peroxisome numbers. This phenotype was accompanied by a strong defect in mitochondrial fission. The MDV1 paralog CAF4 is absent in H. polymorpha. In wild‐type H. polymorpha, cells Dnm1–mCherry and green fluorescent protein (GFP)–Mdv1 colocalize in spots that associate with both peroxisomes and mitochondria. Furthermore, Fis1 is essential to recruit Mdv1 to the peroxisomal and mitochondrial membrane. However, formation of GFP–Mdv1 spots – and related to this normal organelle fission – is strictly dependent on the presence of Dnm1. In dnm1 cells, GFP–Mdv1 is dispersed over the surface of peroxisomes and mitochondria. Also, in H. polymorpha mdv1 or fis1 cells, the number of Dnm1–GFP spots is strongly reduced. These spots still associate to organelles but are functionally inactive.

Pex19p Contributes to Peroxisome Inheritance in the Association of Peroxisomes to Myo2p

During budding of yeast cells peroxisomes are distributed over mother cell and bud, a process that involves the myosin motor protein Myo2p and the peroxisomal membrane protein Inp2p. Here, we show that Pex19p, a peroxin implicated in targeting and complex formation of peroxisomal membrane proteins, also plays a role in peroxisome partitioning. Binding studies revealed that Pex19p interacts with the cargo‐binding domain of Myo2p. We identified mutations in Myo2p that specifically reduced binding to Pex19p, but not to Inp2p. The interaction between Myo2p and Pex19p was also reduced by a mutation that blocked Pex19p farnesylation. Microscopy revealed that the Pex19p–Myo2p interaction is important for peroxisome inheritance, because mutations that affect this interaction hamper peroxisome inheritance in vivo. Together these data suggest that both Inp2p and Pex19p are required for proper association of peroxisomes to Myo2p.

p24 proteins play a role in peroxisome proliferation in yeast

Emp24 is a member of the p24 protein family, which was initially localized to the endoplasmic reticulum, Golgi and COP vesicles, but has recently shown to be associated with Saccharomyces cerevisiae peroxisomes as well. Using cell fractionation and electron‐ and fluorescence microscopy, we show that in the yeast Hansenula polymorpha, Emp24 also associates with peroxisomes. In addition, we show that peroxisome numbers are strongly decreased in H. polymorpha cells lacking two proteins of the p24 complex, Emp24 and Erp3. Detailed fluorescence microscopy analyses suggest that emp24.erp3 cells are disturbed in peroxisome fission and inheritance.

Computational redesign of enzymes for regio- and enantioselective hydroamination

Introduction of innovative biocatalytic processes offers great promise for applications in green chemistry. However, owing to limited catalytic performance, the enzymes harvested from natures biodiversity often need to be improved for their desired functions by time-consuming iterative rounds of laboratory evolution. Here we describe the use of structure-based computational enzyme design to convert Bacillus sp. YM55-1 aspartase, an enzyme with a very narrow substrate scope, to a set of complementary hydroamination biocatalysts. The redesigned enzymes catalyze asymmetric addition of ammonia to substituted acrylates, affording enantiopure aliphatic, polar and aromatic β-amino acids that are valuable building blocks for the synthesis of pharmaceuticals and bioactive compounds. Without a requirement for further optimization by laboratory evolution, the redesigned enzymes exhibit substrate tolerance up to a concentration of 300 g/L, conversion up to 99%, β-regioselectivity >99% and product enantiomeric excess >99%. The results highlight the use of computational design to rapidly adapt an enzyme to industrially viable reactions.Computational protein design, without subsequent directed evolution, rapidly provides a set of aspartase variants capable of biocatalytic asymmetric addition of ammonia to substituted acrylates, producing various enantiopure β-amino acids.

Characterization of the caprolactam degradation pathway in Pseudomonas jessenii using mass spectrometry-based proteomics

Some bacterial cultures are capable of growth on caprolactam as sole carbon and nitrogen source, but the enzymes of the catabolic pathway have not been described. We isolated a caprolactam-degrading strain of Pseudomonas jessenii from soil and identified proteins and genes putatively involved in caprolactam metabolism using quantitative mass spectrometry-based proteomics. This led to the discovery of a caprolactamase and an aminotransferase that are involved in the initial steps of caprolactam conversion. Additionally, various proteins were identified that likely are involved in later steps of the pathway. The caprolactamase consists of two subunits and demonstrated high sequence identity to the 5-oxoprolinases. Escherichia coli cells expressing this caprolactamase did not convert 5-oxoproline but were able to hydrolyze caprolactam to form 6-aminocaproic acid in an ATP-dependent manner. Characterization of the aminotransferase revealed that the enzyme deaminates 6-aminocaproic acid to produce 6-oxohexanoate with pyruvate as amino acceptor. The amino acid sequence of the aminotransferase showed high similarity to subgroup II ω-aminotransferases of the PLP-fold type I proteins. Finally, analyses of the genome sequence revealed the presence of a caprolactam catabolism gene cluster comprising a set of genes involved in the conversion of caprolactam to adipate.

Metabolism of β-valine via a CoA-dependent ammonia lyase pathway

Pseudomonas species strain SBV1 can rapidly grow on medium containing β-valine as a sole nitrogen source. The tertiary amine feature of β-valine prevents direct deamination reactions catalyzed by aminotransferases, amino acid dehydrogenases, and amino acid oxidases. However, lyase- or aminomutase-mediated conversions would be possible. To identify enzymes involved in the degradation of β-valine, a PsSBV1 gene library was prepared and used to complement the β-valine growth deficiency of a closely related Pseudomonas strain. This resulted in the identification of a gene encoding β-valinyl-coenzyme A ligase (BvaA) and two genes encoding β-valinyl-CoA ammonia lyases (BvaB1 and BvaB2). The BvaA protein demonstrated high sequence identity to several known phenylacetate CoA ligases. Purified BvaA enzyme did not convert phenyl acetic acid but was able to activate β-valine in an adenosine triphosphate (ATP)- and CoA-dependent manner. The substrate range of the enzyme appears to be narrow, converting only β-valine and to a lesser extent, 3-aminobutyrate and β-alanine. Characterization of BvaB1 and BvaB2 revealed that both enzymes were able to deaminate β-valinyl-CoA to produce 3-methylcrotonyl-CoA, a common intermediate in the leucine degradation pathway. Interestingly, BvaB1 and BvaB2 demonstrated no significant sequence identity to known CoA-dependent ammonia lyases, suggesting they belong to a new family of enzymes. BLAST searches revealed that BvaB1 and BvaB2 show high sequence identity to each other and to several enoyl-CoA hydratases, a class of enzymes that catalyze a similar reaction with water instead of amine as the leaving group.