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During the microbial degradation of borneol, a bicyclic plant monoterpene, it is first converted into camphor by borneol dehydrogenase (BDH) and then enters a known camphor‐degradation pathway. Previously, a recombinant Pseudomonas BDH was found in inclusion bodies when expressed in Escherichia coli. After refolding, it was still unstable and was difficult to concentrate. Here, the protein‐expression conditions were improved by changing the medium from lysogeny broth to Terrific Broth, yielding a soluble form of the enzyme with higher activity. The protein was crystallized and its 3D structure was determined by X‐ray diffraction. Like other known homologues such as quinuclidinone reductase, the protein forms a tetramer with subunits containing Rossmann folds. Structural comparison revealed major differences in the C‐terminal helices and the associated loops. It is likely that these regions contain the determinants for substrate recognition. [ABSTRACT FROM AUTHOR]

Decaprenyl diphosphate synthase from Mycobacterium tuberculosis (MtDPPS, also known as Rv2361c) catalyzes the consecutive elongation of ω,E,Z‐farnesyl diphosphate (EZ‐FPP) by seven isoprene units by forming new cis double bonds. The protein folds into a butterfly‐like homodimer like most other cis‐type prenyltransferases. The starting allylic substrate EZ‐FPP is bound to the S1 site and the homoallylic substrate to be incorporated, isopentenyl diphosphate, is bound to the S2 site. Here, a 1.55 Å resolution structure of MtDPPS in complex with the substrate analogues geranyl S‐thiodiphosphate (GSPP) and isopentenyl S‐thiodiphosphate bound to their respective sites in one subunit clearly shows the active‐site configuration and the magnesium‐coordinated geometry for catalysis. The ligand‐binding mode of GSPP in the other subunit indicates a possible pathway of product translocation from the S2 site to the S1 site, as required for the next step of the reaction. The preferred binding of negatively charged effectors to the S1 site also suggests a promising direction for inhibitor design. The structure of a complex of Mycobacterium tuberculosis decaprenyl diphosphate synthase with geranyl S‐thiodiphosphate and isopentenyl S‐thiodiphosphate was refined to 1.55 Å resolution. It not only shows the magnesium‐coordinated configuration for catalysis but also suggests a pathway for product translocation as well as a direction for inhibitor design. [ABSTRACT FROM AUTHOR]

Undecaprenyl pyrophosphate (UPP) is an important carrier of the oligosaccharide component in peptidoglycan synthesis. Inhibition of UPP synthase (UPPS) may be an effective strategy in combating the pathogen Acinetobacter baumannii, which has evolved to be multidrug‐resistant. Here, A. baumannii UPPS (AbUPPS) was cloned, expressed, purified and crystallized, and its structure was determined by X‐ray diffraction. Each chain of the dimeric protein folds into a central β‐sheet with several surrounding α‐helices, including one at the C‐terminus. In the active site, two molecules of citrate interact with the side chains of the catalytic aspartate and serine. These observations may provide a structural basis for inhibitor design against AbUPPS. The inhibition of undecaprenyl pyrophosphate synthase (UPPS) may be an effective strategy in combating the multidrug‐resistant pathogen Acinetobacter baumannii. The structure of UPPS from A. baumannii reported here may provide a structural basis for inhibitor design. [ABSTRACT FROM AUTHOR]

S-Adenosylhomocysteine hydrolase (SAHH) catalyzes the reversible conversion of S-adenosylhomocysteine into adenosine and homocysteine. The SAHH from Thermotoga maritima ( TmSAHH) was expressed in Escherichia coli and the recombinant protein was purified and crystallized. TmSAHH crystals belonging to space group C2, with unit-cell parameters a = 106.3, b = 112.0, c = 164.9 Å, β = 103.5°, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.85 Å resolution. Initial phase determination by molecular replacement clearly indicated that the crystal contains one homotetramer per asymmetric unit. Further refinement of the crystal structure is in progress. [ABSTRACT FROM AUTHOR]

β-1,3-1,4-Glucanases catalyze the specific hydrolysis of internal β-1,4-glycosidic bonds adjacent to the 3- O-substituted glucose residues in mixed-linked β-glucans. The thermophilic glycoside hydrolase CtGlu16A from Clostridium thermocellum exhibits superior thermal profiles, high specific activity and broad pH adaptability. Here, the catalytic domain of CtGlu16A was expressed in Escherichia coli, purified and crystallized in the trigonal space group P3121, with unit-cell parameters a = b = 74.5, c = 182.9 Å, by the sitting-drop vapour-diffusion method and diffracted to 1.95 Å resolution. The crystal contains two protein molecules in an asymmetric unit. Further structural determination and refinement are in progress. [ABSTRACT FROM AUTHOR]

The NADH-dependent ( R)-carbonyl reductase from Candida parapsilosis (RCR) catalyzes the asymmetric reduction of 2-hydroxyacetophenone (HAP) to produce ( R)-1-phenyl-1,2-ethanediol [( R)-PED], which is used as a versatile building block for the synthesis of pharmaceuticals and fine chemicals. To gain insight into the catalytic mechanism, the structures of complexes of RCR with ligands, including the coenzyme, are important. Here, the recombinant RCR protein was expressed and purified in Escherichia coli and was crystallized in the presence of NAD+. The crystals, which belonged to the orthorhombic space group P212121, with unit-cell parameters a = 85.64, b = 106.11, c = 145.55 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.15 Å resolution. Initial model building indicates that RCR forms a homotetramer, consistent with previous reports of medium-chain-type alcohol dehydrogenases. [ABSTRACT FROM AUTHOR]

The β-L-arabinofuranosidase (HypBA1) from Bifidobacterium longum JCM 1217 hydrolyzes the β-1,2-linked arabinofuranose disaccharide to release L-arabinoses. HypBA1 was classified into glycoside hydrolase family 127 (GH127) by the CAZy website (). The enzyme was expressed in Escherichia coli and the purified recombinant protein was crystallized. Crystals belonging to the primitive hexagonal space group P3 x21, with unit-cell parameters a = b = 75.9, c = 254.0 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.78 Å resolution. A BLASTP search () of the Protein Data Bank did not reveal any similar crystal structures. Structural determination by using SeMet MAD and MIR methods is in progress. [ABSTRACT FROM AUTHOR]