Henk-Jan Joosten

The alpha/beta-hydrolase fold 3DM database (ABHDB) as a tool for protein engineering.

Aligning the haystack to expose the needle: The 3DM method was used to generate a comprehensive database of the a/s-hydrolase fold enzyme superfamily. This database facilitates the analysis of structure–function relationships and enables novel insights into this superfamily to be made. In addition high-quality libraries for protein engineering can be easily designed.

3DM: Systematic analysis of heterogeneous superfamily data to discover protein functionalities

Ten years of experience with molecular class–specific information systems (MCSIS) such as with the hand‐curated G protein–coupled receptor database (GPCRDB) or the semiautomatically generated nuclear receptor database has made clear that a wide variety of questions can be answered when protein‐related data from many different origins can be flexibly combined. MCSISes revolve around a multiple sequence alignment (MSA) that includes “all” available sequences from the entire superfamily, and it has been shown at many occasions that the quality of these alignments is the most crucial aspect of the MCSIS approach. We describe here a system called 3DM that can automatically build an entire MCSIS. 3DM bases the MSA on a multiple structure alignment, which implies that the availability of a large number of superfamily members with a known three‐dimensional structure is a requirement for 3DM to succeed well. Thirteen MCSISes were constructed and placed on the Internet for examination. These systems have been instrumental in a large series of research projects related to enzyme activity or the understanding and engineering of specificity, protein stability engineering, DNA‐diagnostics, drug design, and so forth. Proteins 2010.

Bioinformatic analysis of a PLP-dependent enzyme superfamily suitable for biocatalytic applications

In this review we analyse structure/sequence-function relationships for the superfamily of PLP-dependent enzymes with special emphasis on class III transaminases. Amine transaminases are highly important for applications in biocatalysis in the synthesis of chiral amines. In addition, other enzyme activities such as racemases or decarboxylases are also discussed. The substrate scope and the ability to accept chemically different types of substrates are shown to be reflected in conserved patterns of amino acids around the active site. These findings are condensed in a sequence-function matrix, which facilitates annotation and identification of biocatalytically relevant enzymes and protein engineering thereof.

Oxaloacetate Hydrolase, the C–C Bond Lyase of Oxalate Secreting Fungi

Oxalate secretion by fungi is known to be associated with fungal pathogenesis. In addition, oxalate toxicity is a concern for the commercial application of fungi in the food and drug industries. Although oxalate is generated through several different biochemical pathways, oxaloacetate acetylhydrolase (OAH)-catalyzed hydrolytic cleavage of oxaloacetate appears to be an especially important route. Below, we report the cloning of the Botrytis cinerea oahA gene and the demonstration that the disruption of this gene results in the loss of oxalate formation. In addition, through complementation we have shown that the intact B. cinerea oahA gene restores oxalate production in an Aspergillus niger mutant strain, lacking a functional oahA gene. These observations clearly indicate that oxalate production in A. niger and B. cinerea is solely dependent on the hydrolytic cleavage of oxaloacetate catalyzed by OAH. In addition, the B. cinera oahA gene was overexpressed in Escherichia coli and the purified OAH was used to define catalytic efficiency, substrate specificity, and metal ion activation. These results are reported along with the discovery of the mechanism-based, tight binding OAH inhibitor 3,3-difluorooxaloacetate (Ki = 68 nm). Finally, we propose that cellular uptake of this inhibitor could reduce oxalate production.

Identification of a Gatekeeper Residue That Prevents Dehydrogenases from Acting as Oxidases

The oxygen reactivity of flavoproteins is poorly understood. Here we show that a single Ala to Gly substitution in l-galactono-γ-lactone dehydrogenase (GALDH) turns the enzyme into a catalytically competent oxidase. GALDH is an aldonolactone oxidoreductase with a vanillyl-alcohol oxidase (VAO) fold. We found that nearly all oxidases in the VAO family contain either a Gly or a Pro at a structurally conserved position near the C4a locus of the isoalloxazine moiety of the flavin, whereas dehydrogenases prefer another residue at this position. Mutation of the corresponding residue in GALDH (Ala-113 → Gly) resulted in a striking 400-fold increase in oxygen reactivity, whereas the cytochrome c reductase activity is retained. The activity of the A113G variant shows a linear dependence on oxygen concentration (kox = 3.5 × 105 m-1 s-1), similar to most other flavoprotein oxidases. The Ala-113 → Gly replacement does not change the reduction potential of the flavin but creates space for molecular oxygen to react with the reduced flavin. In the wild-type enzyme, Ala-113 acts as a gatekeeper, preventing oxygen from accessing the isoalloxazine nucleus. The presence of such an oxygen access gate seems to be a key factor for the prevention of oxidase activity within the VAO family and is absent in members that act as oxidases.

Correlated mutation analyses on super‐family alignments reveal functionally important residues

Correlated mutation analyses (CMA) on multiple sequence alignments are widely used for the prediction of the function of amino acids. The accuracy of CMA‐based predictions is mainly determined by the number of sequences, by their evolutionary distances, and by the quality of the alignments. These criteria are best met in structure‐based sequence alignments of large super‐families. So far, CMA‐techniques have mainly been employed to study the receptor interactions. The present work shows how a novel CMA tool, called Comulator, can be used to determine networks of functionally related residues in enzymes. These analyses provide leads for protein engineering studies that are directed towards modification of enzyme specificity or activity. As proof of concept, Comulator has been applied to four enzyme super‐families: the isocitrate lyase/phoshoenol‐pyruvate mutase super‐family, the hexokinase super‐family, the RmlC‐like cupin super‐family, and the FAD‐linked oxidases super‐family. In each of those cases networks of functionally related residue positions were discovered that upon mutation influenced enzyme specificity and/or activity as predicted. We conclude that CMA is a powerful tool for redesigning enzyme activity and selectivity. Proteins 2009.

Increasing the thermostability of sucrose phosphorylase by a combination of sequence- and structure-based mutagenesis

Sucrose phosphorylase is a promising biocatalyst for the glycosylation of a wide variety of acceptor molecules, but its low thermostability is a serious drawback for industrial applications. In this work, the stability of the enzyme from Bifidobacterium adolescentis has been significantly improved by a combination of smart and rational mutagenesis. The former consists of substituting the most flexible residues with amino acids that occur more frequently at the corresponding positions in related sequences, while the latter is based on a careful inspection of the enzymes crystal structure to promote electrostatic interactions. In this way, a variant enzyme could be created that contains six mutations and whose half-life at the industrially relevant temperature of 60 °C has more than doubled compared with the wild-type enzyme. An increased stability in the presence of organic co-solvents could also be observed, although these effects were most noticeable at low temperatures.

Identification of fungal oxaloacetate hydrolyase within the isocitrate lyase/PEP mutase enzyme superfamily using a sequence marker-based method

Aspergillus niger produces oxalic acid through the hydrolysis of oxaloacetate, catalyzed by the cytoplasmic enzyme oxaloacetate acetylhydrolase (OAH). The A. niger genome encodes four additional open reading frames with strong sequence similarity to OAH yet only the oahA gene encodes OAH activity. OAH and OAH‐like proteins form subclass of the isocitrate lyase/PEP mutase enzyme superfamily, which is ubiquitous present filamentous fungi. Analysis of function‐specific residues using a superfamily‐based approach revealed an active site serine as a possible sequence marker for OAH activity. We propose that presence of this serine in family members correlates with presence of OAH activity whereas its absence correlates with absence of OAH. This hypothesis was tested by carrying out a serine mutagenesis study with the OAH from the fungal oxalic acid producer Botrytis cinerea and the OAH active plant petal death protein as test systems. Proteins 2008.

Novel tools for extraction and validation of disease‐related mutations applied to fabry disease

Genetic disorders are often caused by nonsynonymous nucleotide changes in one or more genes associated with the disease. Specific amino acid changes, however, can lead to large variability of phenotypic expression. For many genetic disorders this results in an increasing amount of publications describing phenotype‐associated mutations in disorder‐related genes. Keeping up with this stream of publications is essential for molecular diagnostics and translational research purposes but often impossible due to time constraints: there are simply too many articles to read. To help solve this problem, we have created Mutator, an automated method to extract mutations from full‐text articles. Extracted mutations are crossreferenced to sequence data and a scoring method is applied to distinguish false‐positives. To analyze stored and new mutation data for their (potential) effect we have developed Validator, a Web‐based tool specifically designed for DNA diagnostics. Fabry disease, a monogenetic gene disorder of the GLA gene, was used as a test case. A structure‐based sequence alignment of the alpha‐amylase superfamily was used to validate results. We have compared our data with existing Fabry mutation data sets obtained from the HGMD and Swiss‐Prot databases. Compared to these data sets, Mutator extracted 30% additional mutations from the literature. Hum Mutat 31:1026–1032, 2010.

Two Subtle Amino Acid Changes in a Transaminase Substantially Enhance or Invert Enantiopreference in Cascade Syntheses

Amine transaminases (ATAs) are powerful enzymes for the stereospecific production of chiral amines. However, the synthesis of amines incorporating more than one stereocenter is still a challenge. We developed a cascade synthesis to access optically active 3‐alkyl‐substituted chiral amines by combining two asymmetric synthesis steps catalyzed by an enoate reductase and ATAs. The ATA wild type from Vibrio fluvialis showed only modest enantioselectivity (14 % de) in the amination of (S)‐3‐methylcyclohexanone, the product of the enoate‐reductase‐catalyzed reaction step. However, by protein engineering we created two variants with substantially improved diastereoselectivities: variant Leu56Val exhibited a higher R selectivity (66 % de) whereas the Leu56Ile substitution caused a switch in enantiopreference to furnish the S‐configured diastereomer (70 % de). Addition of 30 % DMSO further improved the selectivity and facilitated the synthesis of (1R,3S)‐1‐amino‐3‐methylcyclohexane with 89 % de at 87 % conversion.