Characterization of lignin-degrading enzyme PmdC, which catalyzes a key step in the synthesis of polymer precursor 2-pyrone-4,6-dicarboxylic acid.

Pyrone-2,4-dicarboxylic acid (PDC) is a valuable polymer precursor that can be derived from the microbial degradation of lignin. The key enzyme in the microbial production of PDC is 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase, which acts on the substrate CHMS. We present the crystal structure of CHMS dehydrogenase (PmdC from Comamonas testosteroni) bound to the cofactor NADP, shedding light on its three-dimensional architecture, and revealing residues responsible for binding NADP. Using a combination of structural homology, molecular docking, and quantum chemistry calculations, we have predicted the binding site of CHMS. Key histidine residues in a conserved sequence are identified as crucial for binding the hydroxyl group of CHMS and facilitating dehydrogenation with NADP. Mutating these histidine residues results in a loss of enzyme activity, leading to a proposed model for the enzyme's mechanism. These findings are expected to help guide efforts in protein and metabolic engineering to enhance PDC yields in biological routes to polymer feedstock synthesis.

A Synthetic Gene Library Yields a Previously Unknown Glycoside Phosphorylase That Degrades and Assembles Poly-ß-1,3-GlcNAc, Completing the Suite of ß-Linked GlcNAc Polysaccharides.

The considerable utility of glycoside phosphorylases (GPs) has led to substantial efforts over the past two decades to expand the breadth of known GP activities. Driven largely by the increase of available genomic DNA sequence data, the gap between the number of sequences in the carbohydrate active enzyme database (CAZy DB) and its functionally characterized members continues to grow. This wealth of sequence data presented an exciting opportunity to explore the ever-expanding CAZy DB to discover new GPs with never-before-described functionalities. Utilizing an in silico sequence analysis of CAZy family GH94, we discovered and then functionally and structurally characterized the new GP ß-1,3-N-acetylglucosaminide phosphorylase. This new GP was sourced from the genome of the cell-wall-less Mollicute bacterium, Acholeplasma laidlawii and was found to synthesize ß-1,3-linked N-acetylglucosaminide linkages. The resulting poly-ß-1,3-N-acetylglucosamine represents a new, previously undescribed biopolymer that completes the set of possible ß-linked GlcNAc homopolysaccharides together with chitin (ß-1,4) and PNAG (poly-ß-1,6-N-acetylglucosamine). The new biopolymer was denoted acholetin, a combination of the genus Acholeplasma and the polysaccharide chitin, and the new GP was thus denoted acholetin phosphorylase (AchP). Use of the reverse phosphorolysis action of AchP provides an efficient method to enzymatically synthesize acholetin, which is a new biodegradable polymeric material.

Accurate prediction of protein structures and interactions using a three-track neural network.

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.

Author Correction: Jungle Express is a versatile repressor system for tight transcriptional control.

In the original version of this Article, an incorrect URL was provided in the Data Availability Statement regarding the deposition of plasmids listed in Supplementary Table 4. The correct URL is https://public-registry.jbei.org/folders/378 . This error has been corrected in both the PDF and HTML versions of the Article.

Jungle Express is a versatile repressor system for tight transcriptional control.

Tightly regulated promoters are essential for numerous biological applications, where strong inducibility, portability, and scalability are desirable. Current systems are often incompatible with large-scale fermentations due to high inducer costs and strict media requirements. Here, we describe the bottom-up engineering of 'Jungle Express', an expression system that enables efficient gene regulation in diverse proteobacteria. This system is guided by EilR, a multidrug-binding repressor with high affinity to its optimized operator and cationic dyes that act as powerful inducers at negligible costs. In E. coli, the engineered promoters exhibit minimal basal transcription and are inducible over four orders of magnitude by 1 µM crystal violet, reaching expression levels exceeding those of the strongest current bacterial systems. Further, we provide molecular insights into specific interactions of EilR with its operator and with two inducers. The versatility of Jungle Express opens the way for tightly controlled and efficient gene expression that is not restricted to host organism, substrate, or scale.

Engineering glycoside hydrolase stability by the introduction of zinc binding.

The development of robust enzymes, in particular cellulases, is a key step in the success of biological routes to `second-generation' biofuels. The typical sources of the enzymes used to degrade biomass include mesophilic and thermophilic organisms. The endoglucanase J30 from glycoside hydrolase family 9 was originally identified through metagenomic analyses of compost-derived bacterial consortia. These studies, which were tailored to favor growth on targeted feedstocks, have already been shown to identify cellulases with considerable thermal tolerance. The amino-acid sequence of J30 shows comparably low identity to those of previously analyzed enzymes. As an enzyme that combines a well measurable activity with a relatively low optimal temperature (50°C) and a modest thermal tolerance, it offers the potential for structural optimization aimed at increased stability. Here, the crystal structure of wild-type J30 is presented along with that of a designed triple-mutant variant with improved characteristics for industrial applications. Through the introduction of a structural Zn2+ site, the thermal tolerance was increased by more than 10°C and was paralleled by an increase in the catalytic optimum temperature by more than 5°C.

Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes.

Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, co-expression partners, purification methods, and optimization of protein solubility and stability. Hence researchers are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to this process. We devise a pipeline comprising in silico selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification. We have applied this pipeline to a test set of Arabidopsis thaliana cell wall glycosyltransferases known to be challenging to obtain in soluble form, as well as to a library of cell wall glycosyltransferases from other plants including agricultural and biofuel crops. The screening results suggest that recombinant cell wall glycosyltransferases in general have a very low soluble:insoluble ratio in lysates from heterologous expression cultures, and that co-expression of chaperones as well as lysis buffer optimization can increase this ratio. We have applied the identified preferred conditions to Reversibly Glycosylated Polypeptide 1 from Arabidopsis thaliana, and processed this enzyme to near-purity in unprecedented milligram amounts. The obtained preparation of Reversibly Glycosylated Polypeptide 1 has the expected arabinopyranose mutase and autoglycosylation activities.

X-ray diffraction analysis and in vitro characterization of the UAM2 protein from Oryza sativa.

The role of seemingly non-enzymatic proteins in complexes interconverting UDP-arabinopyranose and UDP-arabinofuranose (UDP-arabinosemutases; UAMs) in the plant cytosol remains unknown. To shed light on their function, crystallographic and functional studies of the seemingly non-enzymatic UAM2 protein from Oryza sativa (OsUAM2) were undertaken. Here, X-ray diffraction data are reported, as well as analysis of the oligomeric state in the crystal and in solution. OsUAM2 crystallizes readily but forms highly radiation-sensitive crystals with limited diffraction power, requiring careful low-dose vector data acquisition. Using size-exclusion chromatography, it is shown that the protein is monomeric in solution. Finally, limited proteolysis was employed to demonstrate DTT-enhanced proteolytic digestion, indicating the existence of at least one intramolecular disulfide bridge or, alternatively, a requirement for a structural metal ion.

Structure of a three-domain sesquiterpene synthase: a prospective target for advanced biofuels production.

The sesquiterpene bisabolene was recently identified as a biosynthetic precursor to bisabolane, an advanced biofuel with physicochemical properties similar to those of D2 diesel. High-titer microbial bisabolene production was achieved using Abies grandis α-bisabolene synthase (AgBIS). Here, we report the structure of AgBIS, a three-domain plant sesquiterpene synthase, crystallized in its apo form and bound to five different inhibitors. Structural and biochemical characterization of the AgBIS terpene synthase Class I active site leads us to propose a catalytic mechanism for the cyclization of farnesyl diphosphate into bisabolene via a bisabolyl cation intermediate. Further, we describe the nonfunctional AgBIS Class II active site whose high similarity to bifunctional diterpene synthases makes it an important link in understanding terpene synthase evolution. Practically, the AgBIS crystal structure is important in future protein engineering efforts to increase the microbial production of bisabolene.

Crystal structure of the multidrug efflux transporter AcrB at 3.1A resolution reveals the N-terminal region with conserved amino acids.

Crystal structures of the bacterial multidrug transporter AcrB in R32 and C2 space groups showing both symmetric and asymmetric trimeric assemblies, respectively, supplemented with biochemical investigations, have provided most of the structural basis for a molecular level understanding of the protein structure and mechanisms for substrate uptake and translocation carried out by this 114-kDa inner membrane protein. They suggest that AcrB captures ligands primarily from the periplasm. Substrates can also enter the inner cavity of the transporter from the cytoplasm, but the exact mechanism of this remains undefined. Analysis of the amino acid sequences of AcrB and its homologs revealed the presence of conserved residues at the N-terminus including two phenylalanines which may be exposed to the cytoplasm. Any potential role that these conserved residues may play in function has not been addressed by existing biochemical or structural studies. Since phenylalanine residues elsewhere in the protein have been implicated in ligand binding, we explored the structure of this N-terminal region to investigate structural determinants near the cytoplasmic opening that may mediate drug uptake. Our structure of AcrB in R32 space group reveals an N-terminus loop, reducing the diameter of the central opening to approximately 15 A as opposed to the previously reported value of approximately 30 A for crystal structures in this space group with disordered N-terminus. Recent structures of the AcrB in C2 space group have revealed a helical conformation of this N-terminus but have not discussed its possible implications. We present the crystal structure of AcrB that reveals the structure of the N-terminus containing the conserved residues. We hope that the structural information provides a structural basis for others to design further biochemical investigation of the role of this portion of AcrB in mediating cytoplasmic ligand discrimination and uptake.

Structural genomics of minimal organisms and protein fold space.

The initial aim of the Berkeley Structural Genomics Center is to obtain a near-complete structural complement of two minimal organisms, closely related pathogens Mycoplasma genitalium and M. pneumoniae. The former has fewer than 500 genes and the latter fewer than 700 genes. To achieve this goal, the current protein targets have been selected starting with those predicted to be most tractable and likely to yield new structural and functional information. During the past 3 years, the semi-automated structural genomics pipeline has been set up from cloning, expression, purification, and ultimately to structural determination. The results from the pipeline substantially increased the coverage of the protein fold space of M. pneumoniae and M. genitalium. Furthermore, about 1/2 of the structures of 'unique' protein sequences revealed new and novel folds, and over 2/3 of the structures of previously annotated 'hypothetical proteins' inferred their molecular functions.

Crystal structure of DNA sequence specificity subunit of a type I restriction-modification enzyme and its functional implications.

Type I restriction-modification enzymes are differentiated from type II and type III enzymes by their recognition of two specific dsDNA sequences separated by a given spacer and cleaving DNA randomly away from the recognition sites. They are oligomeric proteins formed by three subunits: a specificity subunit, a methylation subunit, and a restriction subunit. We solved the crystal structure of a specificity subunit from Methanococcus jannaschii at 2.4-A resolution. Two highly conserved regions (CRs) in the middle and at the C terminus form a coiled-coil of long antiparallel alpha-helices. Two target recognition domains form globular structures with almost identical topologies and two separate DNA binding clefts with a modeled DNA helix axis positioned across the CR helices. The structure suggests that the coiled-coil CRs act as a molecular ruler for the separation between two recognized DNA sequences. Furthermore, the relative orientation of the two DNA binding clefts suggests kinking of bound dsDNA and exposing of target adenines from the recognized DNA sequences.