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Abstract: 6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway, catalyzes the oxidative decarboxylation of 6-phosphogluconate, making ribulose 5-phosphate, along with the reduction of NADP+ to NADPH and the release of CO2. Here, we report the first apo-form crystal structure of the pathogenic Klebsiella pneumoniae 6PGDH (Kp6PGDH) and the structures of the highly homologous Escherichia coli K12 6PGDH (Ec6PGDH) complexed with substrate, substrate/NADPH and glucose at high resolution. The binding of NADPH to one subunit of the homodimeric structure triggered a 10° rotation and resulting in a 7Å movement of the coenzyme-binding domain. The coenzyme was thus trapped in a closed enzyme conformation, in contrast to the open conformation of the neighboring subunit. Comparison of our Ec/Kp6PGDH structures with those of other species illustrated how the domain conformation can be affected upon binding of the coenzyme, which in turn gives rise to concomitant movements of two important NADP+-interacting amino acids, M14 and N102. We propose that the catalysis follows an ordered binding mechanism with alternating conformational changes in the corresponding subunits, involving several related amino acid residues. [Copyright &y& Elsevier]

Abstract: Several crystal structures of AFL, a novel lipase from the archaeon Archaeoglobus fulgidus, complexed with various ligands, have been determined at about 1.8 Å resolution. This enzyme has optimal activity in the temperature range of 70–90 °C and pH 10–11. AFL consists of an N-terminal α/β-hydrolase fold domain, a small lid domain, and a C-terminal β-barrel domain. The N-terminal catalytic domain consists of a 6-stranded β-sheet flanked by seven α-helices, four on one side and three on the other side. The C-terminal lipid binding domain consists of a β-sheet of 14 strands and a substrate covering motif on top of the highly hydrophobic substrate binding site. The catalytic triad residues (Ser136, Asp163, and His210) and the residues forming the oxyanion hole (Leu31 and Met137) are in positions similar to those of other lipases. Long-chain lipid is located across the two domains in the AFL–substrate complex. Structural comparison of the catalytic domain of AFL with a homologous lipase from Bacillus subtilis reveals an opposite substrate binding orientation in the two enzymes. AFL has a higher preference toward long-chain substrates whose binding site is provided by a hydrophobic tunnel in the C-terminal domain. The unusually large interacting surface area between the two domains may contribute to thermostability of the enzyme. Two amino acids, Asp61 and Lys101, are identified as hinge residues regulating movement of the lid domain. The hydrogen-bonding pattern associated with these two residues is pH dependent, which may account for the optimal enzyme activity at high pH. Further engineering of this novel lipase with high temperature and alkaline stability will find its use in industrial applications. [Copyright &y& Elsevier]

[Display omitted] • A high-quality cryo-EM structure of malate synthase G (MSG) at 2.89 Å. • A complete structural comparison of a 723-residue MSG determined by cryo-EM and X-ray crystallography. • Cryo-EM revealed the structural dynamics of MSG. • The structural dynamics unraveled by cryo-EM, crystallography and NMR are highly correlated. Determination of sub-100 kDa (kDa) structures by cryo-electron microscopy (EM) is a longstanding but not straightforward goal. Here, we present a 2.9-Å cryo-EM structure of a 723-amino acid apo-form malate synthase G (MSG) from Escherichia coli. The cryo-EM structure of the 82-kDa MSG exhibits the same global folding as structures resolved by crystallography and nuclear magnetic resonance (NMR) spectroscopy, and the crystal and cryo-EM structures are indistinguishable. Analyses of MSG dynamics reveal consistent conformational flexibilities among the three experimental approaches, most notably that the α/β domain exhibits structural heterogeneity. We observed that sidechains of F453, L454, M629, and E630 residues involved in hosting the cofactor acetyl-CoA and substrate rotate differently between the cryo-EM apo-form and complex crystal structures. Our work demonstrates that the cryo-EM technique can be used to determine structures and conformational heterogeneity of sub-100 kDa biomolecules to a quality as high as that obtained from X-ray crystallography and NMR spectroscopy. [ABSTRACT FROM AUTHOR]