Glycosyltransferases are important catalysts for enzymatic and chemoenzymatic synthesis of complex carbohydrates and glycoconjugates. The glycosylation efficiencies of wild-type glycosyltransferases vary considerably when different acceptor substrates were used. Using a multifunctional Pasteurella multocida sialyltransferase 1 (PmST1) as an example, we show here that the sugar nucleotide donor hydrolysis activity of glycosyltransferases contributes significantly to the low yield of glycosylation when a poor acceptor substrate is used. With a protein crystal structure-based rational design, we generated a single mutant (PmST1 M144D) with decreased donor hydrolysis activity without significantly affecting its α2–3-sialylation activity when a poor fucose-containing acceptor substrate was used. The single mutant also has a drastically decreased α2–3-sialidase activity. X-ray and NMR structural studies revealed that unlike the wild-type PmST1 which changes to a closed conformation once a donor binds, the M144D mutant structure adopts an open conformation even in the presence of the donor substrate. The PmST1 M144D mutant with decreased donor hydrolysis and reduced sialidase activity has been used as a powerful catalyst for efficient chemoenzymatic synthesis of complex sialyl Lewisx antigens containing different sialic acid forms. This work sheds new light on the effect of donor hydrolysis activity of glycosyltransferases on glycosyltransferase-catalyzed reactions and provides a novel strategy to improve glycosyltransferase substrate promiscuity by decreasing its donor hydrolysis activity.

Phytochrome photoreceptors control plant growth, development, and the shade avoidance response that limits crop yield in high-density agricultural plantings. Cyanobacteriochromes (CBCRs) are distantly related photosensory proteins that control cyanobacterial metabolism and behavior in response to light. Photoreceptors in both families reversibly photoconvert between two photostates via photoisomerization of linear tetrapyrrole (bilin) chromophores. Spectroscopic and biochemical studies have demonstrated heterogeneity in both photostates, but the structural basis for such heterogeneity remains unclear. We report solution NMR structures for both photostates of the red/green CBCR NpR6012g4 from In addition to identifying structural changes accompanying photoconversion, these structures reveal structural heterogeneity for residues Trp655 and Asp657 in the red-absorbing NpR6012g4 dark state, yielding two distinct environments for the phycocyanobilin chromophore. We use site-directed mutagenesis and fluorescence and absorbance spectroscopy to assign an orange-absorbing population in the NpR6012g4 dark state to the minority configuration for Asp657. This population does not undergo full, productive photoconversion, as shown by time-resolved spectroscopy and absorption spectroscopy at cryogenic temperature. Our studies thus elucidate the spectral and photochemical consequencesof structural heterogeneity in a member of the phytochrome superfamily, insights that should inform efforts to improve photochemical or fluorescence quantum yields in the phytochrome superfamily.

BACKGROUND Neuronal calcium sensor (NCS) proteins, a sub-branch of the calmodulin superfamily, are expressed in the brain and retina where they transduce calcium signals and are genetically linked to degenerative diseases. The amino acid sequences of NCS proteins are highly conserved but their physiological functions are quite distinct. Retinal recoverin and guanylate cyclase activating proteins (GCAPs) both serve as calcium sensors in retinal rod cells, neuronal frequenin (NCS1) modulate synaptic activity and neuronal secretion, K+ channel interacting proteins (KChIPs) regulate ion channels to control neuronal excitability, and DREAM (KChIP3) is a transcriptional repressor that regulates neuronal gene expression. SCOPE OF REVIEW Here we review the molecular structures of myristoylated forms of NCS1, recoverin, and GCAP1 that all look very different, suggesting that the sequestered myristoyl group helps to refold these highly homologous proteins into very different structures. The molecular structure of NCS target complexes have been solved for recoverin bound to rhodopsin kinase, NCS-1 bound to phosphatidylinositol 4-kinase, and KChIP1 bound to A-type K+ channels. MAJOR CONCLUSIONS We propose the idea that N-terminal myristoylation is critical for shaping each NCS family member into a unique structure, which upon Ca2+-induced extrusion of the myristoyl group exposes a unique set of previously masked residues, thereby exposing a distinctive ensemble of hydrophobic residues to associate specifically with a particular physiological target.

Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, confers Ca2+-dependent activation of retinal guanylyl cylcase (RetGC) during phototransduction in vision. Here we analyze the energetics of Ca2+ and Mg2+ binding to the individual EF-hands, characterize metal-induced conformational changes, and evaluate structural effects of myristoylation as studied by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and NMR. GCAP1 binds cooperatively to Ca2+ at EF3 and EF4 (ΔHEF3 = −3.5 kcal/mol and ΔHEF4 = −0.9 kcal/mol) with nanomolar affinity (KEF3 = 80 nM and KEF4 = 200 nM), and a third Ca2+ binds entropically at EF2 (ΔHEF2 = +3.1 kcal/mol and KEF2 = 0.9 μM). GCAP1 binds functionally to Mg2+ at EF2 (ΔHEF2 = +4.3 kcal/mol and KEF2 = 0.7 mM) required for RetGC activation. Ca2+ and/or Mg2+ binding to GCAP1 dramatically alter DSC and NMR spectra, indicating metal-induced protein conformational changes in EF2, EF3 and EF4. Myristoylation of GCAP1 does not significantly alter its metal binding energetics or NMR spectra, suggesting that myristoylation does not influence the structure of the metal-binding EF-hands. Myristoylation also has almost no effect on protein folding stability measured by DSC. NMR resonances of myristate attached to GCAP1 are exchange broadened, upfield shifted and insensitive to Ca2+, consistent with the myristoyl group being sequestered inside the protein as seen in the crystal structure. We conclude that the protein environment near the myristate is not influenced by Mg2+ or Ca2+ binding, but instead is constitutively dynamic and may play a role in promoting GCAP1 interactions with the cyclase.