Richard Haser

Crystal structures of a psychrophilic metalloprotease reveal new insights into catalysis by cold-adapted proteases

Enzymes from psychrophilic organisms differ from their mesophilic counterparts in having a lower thermostability and a higher specific activity at low and moderate temperatures. It is in general accepted that psychrophilic enzymes are more flexible to allow easy accommodation and transformation of the substrates at low energy costs. Here, we report the structures of two crystal forms of the alkaline protease from an Antarctic Pseudomonas species (PAP), solved to 2.1‐ and 1.96‐Å resolution, respectively. Comparative studies of PAP structures with mesophilic counterparts show that the overall structures are similar but that the conformation of the substrate‐free active site in PAP resembles that of the substrate‐bound region of the mesophilic homolog, with both an active‐site tyrosine and a substrate‐binding loop displaying a conformation as in the substrate‐bound form of the mesophilic proteases. Further, a region in the catalytic domain of PAP undergoes a conformational change with a loop movement as large as 13 Å, induced by the binding of an extra calcium ion. Finally, the active site is more accessible due to deletions occurring in surrounding loop regions. Proteins 2003;50:636–647.

Overexpression, purification, crystallization and preliminary diffraction studies of the Protaminobacter rubrum sucrose isomerase SmuA

Palatinose (isomaltulose, alpha-D-glucosylpyranosyl-1,6-D-fructofuranose), a nutritional and acariogenic reducing sugar, is industrially obtained from sucrose by using immobilized cells of Protaminobacter rubrum that produce the sucrose isomerase SmuA. The isomerization of sucrose catalyzed by this enzyme also results in the formation of trehalulose (alpha-D-glucosylpyranosyl-1,1-D-fructofuranose) in smaller amounts and glucose, fructose and eventually isomaltose as by-products, which lower the yield of the reaction and complicate the recovery of palatinose. The determination of the three-dimensional structure of SmuA will provide a basis for rational protein-engineering studies in order to optimize the industrial production of palatinose. A recombinant form of the 67.3 kDa SmuA enzyme has been crystallized in the native state by the vapour-diffusion method. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 61.6, b = 81.4, c = 135.6 A, and diffract to 1.95 A resolution on a synchrotron-radiation source.

Expression, purification, crystallization and preliminary X-ray crystallographic studies of the trehalulose synthase MutB from Pseudomonas mesoacidophila MX-45

The trehalulose synthase (MutB) from Pseudomonas mesoacidophila MX-45, belonging to glycoside hydrolase family 13, catalyses the isomerization of sucrose to trehalulose (alpha-D-glucosylpyranosyl-1,1-D-fructofuranose) and isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofuranose) as main products and glucose and fructose in residual amounts from the hydrolytic reaction. To date, a three-dimensional structure of a sucrose isomerase that produces mainly trehalulose, as is the case for MutB, has been lacking. Crystallographic studies of this 64 kDa enzyme have therefore been initiated in order to contribute to the understanding of the molecular basis of sucrose decomposition, isomerization and of the selectivity of this enzyme that leads to the formation of different products. The MutB protein has been overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. Two different crystal forms have been obtained: one diffracts X-rays to 1.6 A resolution using synchrotron radiation and belongs to space group P1, with unit-cell parameters a = 63.8, b = 72.0, c = 82.2 A, alpha = 67.5, beta = 73.1, gamma = 70.8 degrees, while the other form diffracts to 1.8 A resolution using synchrotron radiation and belongs to space group P2(1), with unit-cell parameters a = 63.7, b = 85.9, c = 119.7 A, beta = 97.7 degrees. A molecular-replacement solution has been found using the structure of the isomaltulose synthase (PalI) from Klebsiella sp. LX3 as a search model.

Crystallization and preliminary X-ray analysis of a bacterial psychrophilic enzyme, phosphoglycerate kinase

The glycolytic enzyme phosphoglycerate kinase (PGK) from the Antarctic microorganism Pseudomonas sp. TACII18 is a cold-adapted enzyme that displays a high specific activity at low temperatures and decreased thermostability relative to its mesophilic counterpart. Herein, the preliminary crystallization and structure solution of psychrophilic PGK in its native form and cocrystallized with 3-phosphoglyceric acid (3-PGA) and the ATP analogue adenylyl imidophosphate (AMP-PNP) is reported. The complexed form of PGK crystallized in 2-3 d at 290 K, whereas the native form of the enzyme required 8-12 months. Morphologically, both crystal forms are similar and X-ray diffraction experiments indicate that the crystals are isomorphous. The crystals diffracted to a resolution of 2.0 A and belong to the space group P3(2). with unit-cell parameters a = b = 58.5, c = 85.4 A.

Crystallization and preliminary X-ray study of an N-­terminal fragment of rat liver ribosomal P2 protein

Ribosomal P proteins have been shown to be involved in the binding of elongation factors and participate in factor-dependent GTP hydrolysis. The P proteins form the pentamer (P1/P2)(2)-P0 constituting the lateral flexible stalk of the 60S ribosomal subunit. The highly soluble domain (1-65) of rat liver P2 has been overexpressed in Escherichia coli as an N-terminal poly-His-tagged protein and crystallized. To reduce nucleation and improve crystal morphology and diffraction power, the crystals were grown in a gel matrix and an oil barrier was added between the reservoir and the drop to reduce the rate of vapour diffusion. This dramatically reduced the nucleation in the drops and yielded diffraction-quality crystals. Data were collected to 2.4 A resolution at beamline ID 14-1, ESRF. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 37.7, b = 96.7, c = 135.0 A.