An α2,3-Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution.

Defined sialoglycoconjugates are important molecular probes for studying the role of sialylated glycans in biological systems. We show that the α2,3-sialyltransferase from Photobacterium phosphoreum JT-ISH-467 (2,3SiaTpph ) tolerates a very broad substrate scope for modifications in the sialic acid part, including bulky amide variation, C5/C9 substitution, and C5 stereoinversion. To reduce the enzyme's hydrolytic activity, which erodes the product yield, an extensive structure-guided mutagenesis study identified three variants that show up to five times higher catalytic efficiency for sialyltransfer, up to ten times lower efficiency for substrate hydrolysis, and drastically reduced product hydrolysis. Variant 2,3SiaTpph (A151D) displayed the best performance overall in the synthesis of the GM3 trisaccharide (α2,3-Neu5Ac-Lac) from lactose in a one-pot, two-enzyme cascade. Our study demonstrates that several complementary solutions can be found to suppress the common problem of undesired hydrolysis activity of microbial GT80 sialyltransferases. The new enzymes are powerful catalysts for the synthesis of a wide variety of complex natural and new-to-nature sialoconjugates for biological studies.

Donor Promiscuity of a Thermostable Transketolase by Directed Evolution: Efficient Complementation of 1-Deoxy-d-xylulose-5-phosphate Synthase Activity.

Enzymes catalyzing asymmetric carboligation reactions typically show very high substrate specificity for their nucleophilic donor substrate components. Structure-guided engineering of the thermostable transketolase from Geobacillus stearothermophilus by directed in vitro evolution yielded new enzyme variants that are able to utilize pyruvate and higher aliphatic homologues as nucleophilic components for acyl transfer instead of the natural polyhydroxylated ketose phosphates or hydroxypyruvate. The single mutant H102T proved the best hit toward 3-methyl-2-oxobutyrate as donor, while the double variant H102L/H474S showed highest catalytic efficiency toward pyruvate as donor. The latter variant was able to complement the auxotrophic deficiency of Escherichia coli cells arising from a deletion of the dxs gene, which encodes for activity of the first committed step into the terpenoid biosynthesis, offering the chance to employ a growth selection test for further enzyme optimization.

The interaction of ETV6 (TEL) and TIP60 requires a functional histone acetyltransferase domain in TIP60.

The ets-family transcription factor ETV6 (TEL) has been shown to be the target of a large number of balanced chromosomal translocations in various hematological malignancies and in some soft tissue tumors. Furthermore, ETV6 is essential for hematopoietic stem cell function. We identified ETV6 interacting proteins using the yeast two hybrid system. One of these proteins is the HIV Tat interacting protein (TIP60), a histone acetyltransferase (HAT) containing the highly conserved MYST domain. TIP60 functions as a corepressor of ETV6 in reporter gene assays. Fluorescently tagged ETV6 and TIP60 colocalize in the nucleus and an increase in nuclear localization of ETV6 was seen when TIP60 was cotransfected. ETV6 interacts with TIP60 through a 63 amino acids region located in the central domain of ETV6 between the pointed and the ets domain. The ETV6 interacting region of TIP60 mapped to the C2HC zinc finger of the TIP60 MYST domain. The interaction of TIP60 with full length ETV6 required an intact acetyltransferase domain of TIP60. Interestingly, the MYST domains of MOZ and MORF were also able to interact with portions of ETV6. These observations suggest that MYST domain HATs regulate ETV6 transcriptional activity and may therefore play critical roles in leukemogenesis and possibly in normal hematopoietic development.