Search

In metabolic engineering and synthetic biology fields, there have been efforts to produce variable bioalcohol fuels, such as isobutanol and 2-phenylethanol, in order to meet industrial demands. YjgB is an aldehyde dehydrogenase from Escherichia coli that shows nicotinamide adenine dinucleotide phosphate (NADP)-dependent broad selectivity for aldehyde derivatives with an aromatic ring or small aliphatic chain. This could contribute to the design of industrial synthetic pathways. We determined the crystal structures of YjgB for both its apo-form and NADP-complexed form at resolutions of 1.55 and 2.00 Å, respectively, in order to understand the mechanism of broad substrate selectivity. The hydrophobic pocket of the active site and the nicotinamide ring of NADP(H) are both involved in conferring its broad specificity toward aldehyde substrates. In addition, based on docking-simulation data, we inferred that π-π stacking between substrates and aromatic side chains might play a crucial role in recognizing substrates. Our structural analysis of YjgB might provide insights into establishing frameworks to understand its broad substrate specificity and develop engineered enzymes for industrial biofuel synthesis.

YgjG is a putrescine aminotransferase enzyme that transfers amino groups from compounds with terminal primary amines to compounds with an aldehyde group using pyridoxal-5'-phosphate (PLP) as a cofactor. Previous biochemical data show that the enzyme prefers primary diamines, such as putrescine, over ornithine as a substrate. To better understand the enzyme's substrate specificity, crystal structures of YgjG from Escherichia coli were determined at 2.3 and 2.1 Å resolutions for the free and putrescine-bound enzymes, respectively. Sequence and structural analyses revealed that YgjG forms a dimer that adopts a class III PLP-dependent aminotransferase fold. A structural comparison between YgjG and other class III aminotransferases revealed that their structures are similar. However, YgjG has an additional N-terminal helical structure that partially contributes to a dimeric interaction with the other subunit via a helix-helix interaction. Interestingly, the YgjG substrate-binding site entrance size and charge distribution are smaller and more hydrophobic than other class III aminotransferases, which suggest that YgjG has a unique substrate binding site that could accommodate primary aliphatic diamine substrates, including putrescine. The YgjG crystal structures provide structural clues to putrescine aminotransferase substrate specificity and binding.

Penicillin-binding proteins (PBPs), which mediate the peptidoglycan biosynthetic pathway in the bacterial cell wall, have been intensively investigated as a target for the design of antibiotics. In this study, PBPD2, a low-molecular-weight PBP encoded by lmo2812 from Listeria monocytogenes, was overexpressed in Escherichia coli, purified and crystallized at 295 K using the sitting-drop vapour-diffusion method. The crystal belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 37.7, b = 74.7, c = 75.1 Å, and diffracted to 1.55 Å resolution. There was one molecule in the asymmetric unit. The preliminary structure was determined by the molecular-replacement method.

Putrescine, one of the polyamines that are found in virtually all living organisms, has been implicated as an important biological material. The protein YgjG is involved in the putrescine-degradation pathway in Escherichia coli. The enzyme is a putrescine:2-oxoglutarate aminotransferase that belongs to the class III aminotransferases. In this study, YgjG from E. coli was overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected to 2.1 Å resolution using synchrotron radiation. The crystal belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 121.1, b = 129.5, c = 131.3 Å, and is estimated to contain four molecules of YgjG per asymmetric unit.

SSADH is involved in the final step of GABA degradation, converting SSA to succinic acid in the human mitochondrial matrix, and its activity is known to be regulated via 'redox-switch modulation' of the catalytic loop. We present the crystal structure of EcSSADH, revealing that the catalytic loop of EcSSADH, unlike that of human SSADH, does not undergo disulfide bond-mediated structural changes upon changes of environmental redox status. Subsequent redox change experiments using recombinant proteins confirm the non-redox regulation of this protein. Detailed structural analysis shows that a difference in the conformation of the connecting loop (beta15-beta16) causes the formation of a water molecule-mediated hydrogen bond network between the connecting loop and the catalytic loop in EcSSADH, making the catalytic loop of EcSSADH more rigid compared to that of human SSADH. The cytosolic localization of EcSSADH and the cellular function of the GABA shunt in E. coli might result in the non-redox mediated regulatory mechanisms of the protein., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

MazG is a nucleoside triphosphate pyrophosphohydrolase that hydrolyzes all canonical nucleoside triphosphates. The mazG gene located downstream from the chromosomal mazEF "addiction module," that mediated programmed cell death in Escherichia coli. MazG activity is inhibited by the MazEF complex both in vivo and in vitro. Enzymatic activity of MazG in vivo affects the cellular level of guanosine 3',5'-bispyrophosphate (ppGpp), synthesized by RelA under amino acid starvation. The reduction of ppGpp, caused by MazG, may extend the period of cell survival under nutritional stress. Here we describe the first crystal structure of active MazG from E. coli, which is composed of two similarly folded globular domains in tandem. Among the two putative catalytic domains, only the C-terminal domain has well ordered active sites and exhibits an NTPase activity. The MazG-ATP complex structure and subsequent mutagenesis studies explain the peculiar active site environment accommodating all eight canonical NTPs as substrates. In vivo nutrient starvation experiments show that the C terminus NTPase activity is responsible for the regulation of bacterial cell survival under nutritional stress.

We report the crystal structure of PYCH_01220, a hypothetical protein in Pyrococcus yayanosii CH1. This protein is composed of two domains, named Domain A and Domain B. While Domain B is not significantly homologous to known protein structures, Domain A is structurally analogous to the C‐terminal ribonuclease domain of Escherichia coli colicin D. Domain A has a positively charged surface patch rendered by 13 basic residues, eight arginine or lysine residues of which are evolutionarily conserved. Electrophoretic mobility shift assays showed that PYCH_01220 binds to DNA, and charge‐inversion mutations on this patch negatively affect the DNA binding, suggesting that the function of PYCH_01220 might involve nucleic acid‐binding via the positively charged patch. [ABSTRACT FROM AUTHOR]

Abstract: Lysine biosynthesis has been of interest in plant research, because lysine is the most limiting amino acid in crop protein production. Dihydrodipicolinate synthase (DHDPS) catalyzes the branch point reaction leading to meso-diaminopimelate (DAP) and (S)-lysine in lysine biosynthesis. In this report, we present the crystal structure of DHDPS from the marine bacterium Hahella chejuensis (HcDHDPS) at 1.5Å resolution. The four subunits of the asymmetric unit assemble to form a tetramer with an approximate 222 symmetry. At the active site of HcDHDPS, three residues Tyr132, Thr43 and Tyr106 are observed to constitute a catalytic triad and are located at similar positions of the corresponding residues of Escherichia coli DHDPS. The structural similarities in the overall fold and the active site environment between these two enzymes imply that HcDHDPS functions by a mechanism similar to E. coli DHDPS. However, unlike E. coli DHDPS, HcDHDPS has a unique extensive dimer–dimer interface that is mediated by not only strong hydrophobic interactions but also a hydrogen bond network. [Copyright &y& Elsevier]