Alastair R. Hawkins

Structure of dehydroquinate synthase reveals an active site capable of multistep catalysis

Dehydroquinate synthase (DHQS) has long been regarded as a catalytic marvel because of its ability to perform several consecutive chemical reactions in one active site. There has been considerable debate as to whether DHQS is actively involved in all these steps,, or whether several steps occur spontaneously, making DHQS a spectator in its own mechanism. DHQS performs the second step in the shikimate pathway, which is required for the synthesis of aromatic compounds in bacteria, microbial eukaryotes and plants. This enzyme is a potential target for new antifungal and antibacterial drugs, as the shikimate pathway is absent from mammals and DHQS is required for pathogen virulence. Here we report the crystal structure of DHQS, which has several unexpected features, including a previously unobserved mode for NAD+-binding and an active-site organization that is surprisingly similar to that of alcohol dehydrogenase, in a new protein fold. The structure reveals interactions between the active site and a substrate-analogue inhibitor, which indicate how DHQS can perform multistep catalysis without the formation of unwanted by-products.

The two types of 3-dehydroquinase have distinct structures but catalyze the same overall reaction.

The structures of enzymes catalyzing the reactions in central metabolic pathways are generally well conserved as are their catalytic mechanisms. The two types of 3-dehydroquinate dehydratase (DHQase) are therefore most unusual since they are unrelated at the sequence level and they utilize completely different mechanisms to catalyze the same overall reaction. The type I enzymes catalyze a cis-dehydration of 3-dehydroquinate via a covalent imine intermediate, while the type II enzymes catalyze a trans-dehydration via an enolate intermediate. Here we report the three-dimensional structures of a representative member of each type of biosynthetic DHQase. Both enzymes function as part of the shikimate pathway, which is essential in microorganisms and plants for the biosynthesis of aromatic compounds including folate, ubiquinone and the aromatic amino acids. An explanation for the presence of two different enzymes catalyzing the same reaction is presented. The absence of the shikimate pathway in animals makes it an attractive target for antimicrobial agents. The availability of these two structures opens the way for the design of highly specific enzyme inhibitors with potential importance as selective therapeutic agents.

Glycan complexity dictates microbial resource allocation in the large intestine

The structure of the human gut microbiota is controlled primarily through the degradation of complex dietary carbohydrates, but the extent to which carbohydrate breakdown products are shared between members of the microbiota is unclear. We show here, using xylan as a model, that sharing the breakdown products of complex carbohydrates by key members of the microbiota, such as Bacteroides ovatus, is dependent on the complexity of the target glycan. Characterization of the extensive xylan degrading apparatus expressed by B. ovatus reveals that the breakdown of the polysaccharide by the human gut microbiota is significantly more complex than previous models suggested, which were based on the deconstruction of xylans containing limited monosaccharide side chains. Our report presents a highly complex and dynamic xylan degrading apparatus that is fine-tuned to recognize the different forms of the polysaccharide presented to the human gut microbiota.

The structure of the negative transcriptional regulator NmrA reveals a structural superfamily which includes the short‐chain dehydrogenase/reductases

NmrA is a negative transcriptional regulator involved in the post‐translational modulation of the GATA‐type transcription factor AreA, forming part of a system controlling nitrogen metabolite repression in various fungi. X‐ray structures of two NmrA crystal forms, both to 1.8 Å resolution, show NmrA consists of two domains, including a Rossmann fold. NmrA shows an unexpected similarity to the short‐chain dehydrogenase/reductase (SDR) family, with the closest relationship to UDP‐galactose 4‐epimerase. We show that NAD binds to NmrA, a previously unreported nucleotide binding property for this protein. NmrA is unlikely to be an active dehydrogenase, however, as the conserved catalytic tyrosine in SDRs is absent in NmrA, and thus the nucleotide binding to NmrA could have a regulatory function. Our results suggest that other transcription factors possess the SDR fold with functions including RNA binding. The SDR fold appears to have been adapted for other roles including non‐enzymatic control functions such as transcriptional regulation and is likely to be more widespread than previously recognized.

The Mycobacterium tuberculosis shikimate pathway genes : evolutionary relationship between biosynthetic and catabolic 3-dehydroquinases

SummaryThe Mycobacterium tuberculosis shikimate pathway genes designated aroB and aroQ encoding 3-dehydroquinate synthase and 3-dehydroquinase, respectively were isolated by molecular cloning and their nucleotide sequences determined. The deduced dehydroquinate synthase amino acid sequence from M. tuberculosis showed high similarity to those of equivalent enzymes from prokaryotes and filamentous fungi. Surprisingly, the deduced M. tuberculosis 3-dehydroquinase amino acid sequence showed no similarity to other characterised prokaryotic biosynthetic 3-dehydroquinases (bDHQases). A high degree of similarity was observed, however, to the fungal catabolic 3-dehydroquinases (cDHQases) which are active in the quinic acid utilisation pathway and are isozymes of the fungal bDHQases. This finding indicates a common ancestral origin for genes encoding the catabolic dehydroquinases of fungi and the biosynthetic dehydroquinases present in some prokaryotes. Deletion of genes encoding shikimate pathway enzymes represents a possible approach to generation of rationally attenuated strains of M. tuberculosis for use as live vaccines.

Crystal Structure of SANOS, a Bacterial Nitric Oxide Synthase Oxygenase Protein from Staphylococcus aureus

Prokaryotic genes related to the oxygenase domain of mammalian nitric oxide synthases (NOSs) have recently been identified. Although they catalyze the same reaction as the eukaryotic NOS oxygenase domain, their biological function(s) are unknown. In order to explore rationally the biochemistry and evolution of the prokaryotic NOS family, we have determined the crystal structure of SANOS, from methicillin-resistant Staphylococcus aureus (MRSA), to 2.4 A. Haem and S-ethylisothiourea (SEITU) are bound at the SANOS active site, while the intersubunit site, occupied by the redox cofactor tetrahydrobiopterin (H(4)B) in mammalian NOSs, has NAD(+) bound in SANOS. In common with all bacterial NOSs, SANOS lacks the N-terminal extension responsible for stable dimerization in mammalian isoforms, but has alternative interactions to promote dimer formation.

Molecular organisation of the quinic acid utilization (QUT) gene cluster in Aspergillus nidulans

SummaryThe functional integrity of the QUTB gene (encoding quinate dehydrogenase) has been confirmed by transformation of a qutB mutant strain. The DNA sequence of the contiguous genes QUTD (quinate permease), QUTB and QUTG (function unknown) has been determined and analysed, together with that of QUTE (catabolic 3-dehydroquinase). The QUTB sequence shows significant homology with the shikimate dehydrogenase function of the complex AROM locus of Aspergillus nidulans, and with the QA-3 quinate dehydrogenase and QA-1S (repressor) genes of Neurospora crassa. The QUTD gene shows strong homology with the N. crassa QA-Y gene and QUTG with the QA-X gene. QUTD, QUTB, and QUTG, QUTE form two pairs of divergently transcribed genes, and conserved sequence motifs identified in the two common 5′ non-coding regions show significant homology with UASGAL and UASQA sequences of the Saccharomyces cerevisiae and N. crassa Gal and QA systems. In addition, conserved 5′ sequences homologous to the mammalian CAAT box are noted and a previously unreported conserved 22 nucleotide motif is presented.

Actin in the oomycetous fungus Phytophthora infestans is the product of several genes.

Actin (ACT) in Phytophthora infestans is encoded by at least two genes, in contrast to unicellular and other filamentous fungi where there is a single gene. These genes (designated actA and actB) have been isolated from a genomic library of P. infestans. The complete nucleotide sequence of both genes has been determined. Unlike the actin-encoding genes (act) of other filamentous fungi, no introns are obvious in the coding region, a feature shared with the act genes of certain protists. Northern blotting and primer extension studies of the mRNA show that actA and actB are actively transcribed in mycelium, sporangia and germinating cysts but only at a low level in the case of actB. Both genes display bias in their codon usage. This is more extreme in actA. The deduced ACTB protein is strikingly similar to that of the Phytophthora megasperma actin and is more diverged from other actins than ACTA.

Structures of R- and T-state Escherichia coli Aspartokinase III MECHANISMS OF THE ALLOSTERIC TRANSITION AND INHIBITION BY LYSINE

Aspartokinase III (AKIII) from Escherichia coli catalyzes an initial commitment step of the aspartate pathway, giving biosynthesis of certain amino acids including lysine. We report crystal structures of AKIII in the inactive T-state with bound feedback allosteric inhibitor lysine and in the R-state with aspartate and ADP. The structures reveal an unusual configuration for the regulatory ACT domains, in which ACT2 is inserted into ACT1 rather than the expected tandem repeat. Comparison of R- and T-state AKIII indicates that binding of lysine to the regulatory ACT1 domain in R-state AKIII instigates a series of changes that release a “latch”, the β15-αK loop, from the catalytic domain, which in turn undergoes large rotational rearrangements, promoting tetramer formation and completion of the transition to the T-state. Lysine-induced allosteric transition in AKIII involves both destabilizing the R-state and stabilizing the T-state tetramer. Rearrangement of the catalytic domain blocks the ATP-binding site, which is therefore the structural basis for allosteric inhibition of AKIII by lysine.

The Aspergillus niger niaD gene encoding nitrate reductase: upstream nucleotide and amino acid sequence comparisons

The Aspergillus niger niaD gene has been sequenced and the inferred nitrate reductase (NR) protein found to consist of 867 amino acid residues (97 kDa). The gene is interrupted by six small introns, as deduced by comparison with the niaD gene of Aspergillus nidulans. The positions of these putative introns are conserved between the two fungi, although the sequences are dissimilar. The niiA gene, encoding nitrite reductase, the second reductive step in the nitrate assimilation pathway, is tightly linked to niaD and divergently transcribed in A. niger, similar to the general organisation in the related fungi, Aspergillus oryzae and A. nidulans. The nucleotide (nt) sequences of the intergenic region between niiA and niaD (excluding the ATG translation start codon) of A. niger (1668 nt) and A. oryzae (1575 nt) were determined and compared with the previously determined A. nidulans (1262 nt) sequence. No striking extended nt regions of homology are observed in spite of the fact that transgenic strains with fungal niaD or the two control genes required for induction and repression show virtually normal regulation. Fungal NR shows considerable aa homology with higher plant NR, particularly within the co-factor domains for flavin adenoside dinucleotide, heme and molybdopterin cofactor.