Research Programs in the BMB Department

Research Programs
Graduate Program in Biochemistry and Molecular Biology

Chemistry of Copper-Containing Enzymes

Increasing numbers of important enzymes are known to contain copper at their active sites. Of particular interest are enzymes involved in biogenic amine biosynthesis and metabolism (including important neuroactive amines such as nor-adrenaline and amphetamine); enzymes protecting against oxidative cellular damage caused by reduced oxygen metabolites; and enzymes catalyzing the biosynthesis of neuropeptide hormones. A major goal is to understand the catalytic role of copper and the molecular mechanism of oxygen binding and utilization by these oxidase and oxygenase enzymes. Blackburn

Spectroscopy of Copper Proteins

Spectroscopic techniques are used to probe the structures of the copper sites in the native proteins and their complexes with substrates and inhibitors. Since the chemistry of the catalytic processes is generally centered on the Cu(I) forms of the enzymes, we are concentrating on the challenging task of developing spectroscopic probes of the Cu(I) oxidation state, which is transparent to most common spectroscopic techniques. Our work thus includes Fourier transform infrared, X-ray absorption edge, and EXAFS spectroscopies, and emphasizes the use of computer simulation of spectra on our micro SGI Indigo work station. Data for the latter two techniques are collected at national and international synchrotron radiation facilities. Proteins under investigation include dopamine-ß hydroxylase, cytochrome c oxidase, hemocyanin, peptide amidating enzyme, Menkes and Wilson proteins, and copper chaperones. Blackburn

Anaerobiosis of Bacillus subtilis

A gram-positive soil bacterium B. subtilis is highly amenable to genetic analysis and has been used as a model system to study fundamental microbiological research. In addition, B. subtilis is medically and industrially important since it produces a variety of antibiotics and extracellular enzymes. Although the organism has been widely used, it has been mistakenly referred to as a strict aerobe until recently. Our studies, together with others, have shown that B. subtilis is able to grow under anaerobic conditions by utilizing nitrate or nitrite as an alternative electron acceptor. In the absence of terminal electron acceptors, it undergoes fermentative growth. Our research aims include elucidation of the regulatory mechanisms through which the cells adapt to oxygen limitation. Molecular genetic and biochemical approaches are applied. Nakano

Two Physiological Roles of Nitrate and Nitrite Reductases

Nitrate and nitrite reductases have two roles in metabolism of B. subtilis: assimilation of nitrate/ nitrite and anaerobic respiration. Two genetically- and biochemically distinct nitrate reductases are present to fulfill the dual roles; in contrast, a single nitrite reductase functions in both assimilation and respiration. The functional differences of the enzymes correspond to the difference in gene regulation. We have studied how these nitrate and nitrite reductase genes are regulated in response to nitrogen and oxygen limitation by promoter analysis of these genes and identification of trans-acting factors. The mechanisms of transcriptional activation of the nitrate/nitrite reductase genes are being investigated. Nakano

ResD-ResE Two-Component Signal Transduction System

Bacteria often encounter sudden environmental changes. Cells cope with such changes by an elaborate network of adaptive responses. The two-component signal transduction system senses and then processes information derived from environmental changes so that the cell can choose the appropriate adaptive response. This simple signal transduction system is widespread in bacteria and also found in plants and lower eukaryotes. ResE is a histidine kinase and ResD is a response regulator of this large protein family. We have shown that ResD and ResE are indispensable for anaerobic respiration in B. subtilis. A specific signal derived by oxygen limitation is recognized by the N-terminal input domain of the ResE kinase leading to autophosphorylation of a conserved histidine residue in the C-terminal transmitter domain. This phosphoryl group is then transferred to aspartate in the conserved N-terminal domain of ResD, altering the activity of its C-terminal domain as a transcriptional activator. The objectives of our studies are to determine how ResE senses oxygen limitation and how anaerobically induced genes are activated by ResD. Nakano

Flavohemoglobin (Hmp)

Flavohemoglobin is a ubiquitous protein present in organisms ranging from Escherichia coli to Saccharomyces cerevisiae. The N-terminal part of the protein has similarity to hemoglobin and the C-terminus is homologous to reductase with a flavin-binding domain. B. subtilis hmp was identified among genes, expression of which is induced by oxygen limitation. The anaerobic induction of hmp requires the ResD-ResE signal transduction pairs and nitrite. The detailed regulatory mechanism of hmp expression and its functional role in anaerobiosis are under investigation. Nakano

Peptide Antibiotic Biosynthesis

Our research is aimed at understanding the mechanism of antimicrobial peptide biosynthesis. Peptide antibiotics are synthesized either by the non-ribosomal thiotemplate mechanism, or are bacteriocins that are gene-encoded and synthesized on ribosomes. Both classes are used as bio-control agents in medicine, agriculture, and in the food industry. Non-ribosomally synthesized peptides also include iron-scavenging siderophores which are required for virulence by some bacterial pathogens and toxins produced by a variety of bacterial and fungal species that infect plants. A knowledge of how peptide and bacteriocin biosyntheses are carried out at the molecular level may provide information which can ultimately be used to design ways to control the virulence of pathogenic microorganisms and to synthesize peptides with a defined structure and bioactivity. The spore-forming bacterium Bacillus subtilis will produce an abundance of peptide antibiotics and bacteriocins under conditions of nutritional stress and oxygen limitation. The genes encoding the enzymes that catalyze peptide biosynthesis have been cloned and we are now engaged in genetic engineering of the enzymes to understand the mechanism of antimicrobial peptide biosynthesis. Zuber

Prokaryotic Signal Transduction/Gene Regulation

Bacteria can respond in variety of ways to a growth-restricting environment. Prolonged exposure to a nutritionally poor environment results in the induction of antibiotic biosynthesis, functions required for cell motility and processes of cellular differentiation that give rise to highly resistant cell types. How cells respond to nutritional stress is profoundly influenced by cell density. Extracellular signal molecules accumulate in the local environment of densely populated cell cultures and trigger antibiotic production and developmental processes such as sporulation and genetic competence. The objective of our research is to understand, in molecular terms, the regulatory networks that cells utilize to choose the most appropriate response to harsh conditions. In the spore-forming
bacterium, Bacillus subtilis, establishment of genetic competence is co-regulated with peptide antibiotic biosynthesis by a complex network of signal transduction pathways that utilize protein components common to all prokaryotic and most eukaryotic organisms. Zuber

Translational Control of Human Proto-Oncogenes

The transcripts specified by many genes involved in human cancers contain uORFs; these include the her-2 and bcl-2 proto-oncogenes. Using methods similar to those developed for understanding the roles of the uORFs of N. crassa arg-2 and S. cerevisiae CPA1 gene expression, we are examining the functions of these mammalian uORFs, to better understand their role in controlling the expression of these critically important genes. Sachs

The Neurospora Genome

We are part of a team that is sequencing and annotating the genome of Neurospora crassa (see www-genome.wi.mit.edu/annotation/fungi/neurospora/), and are gearing up to apply this information to large-scale community-wide efforts in functional genomics. This is the first genome of a filamentous fungus that has been sequenced with public funds; the annotation of this sequence is proving invaluable for understanding fungal genome evolution; many fungi important for agriculture and medicine are closely related to N. crassa. We recently began experiments aimed at cloning and analyzing the telomeric regions of N. crassa and the closely related pathogenic rice blast fungus Magnaporthe grisea because mounting evidence indicates that genes near telomeres evolve more quickly and are frequently involved in pathogenic interactions with hosts. Sachs

Translational and Transcriptional Control in Neurospora crassa

A greater understanding of many human health issues relies on increased knowledge of how cells express genetic information. Gene expression can be controlled by regulating the synthesis and stability of functional RNA and protein. The goal of our research is to obtain a greater understanding of how these mechanisms work using the Neurospora crassa arg-2 and Saccharomyces cerevisiae CPA1 genes as models. These homologous genes encode the first enzyme in arginine biosynthesis and they are negatively regulated at both transcriptional and translational levels in response to the availability of arginine. An evolutionarily conserved upstream open reading frame (uORF) present in the 5'-leader regions of these transcripts is responsible for translational control. Synthesis of the uORF-encoded peptide causes ribosomes to stall when the level of arginine is high, blocking access of ribosomes to the translation initiation site for the polypeptide encoding the arginine biosynthetic enzyme. Our current work is focused on developing a molecular understanding of how synthesis of this uORF-encoded peptide causes ribosomes to stall, since this will provide important insights into the fundamental cellular process of protein synthesis. Sachs

Mechanisms of Chemosensory Responses in Elephants and Sharks

Temporal sampling Chemical communication plays a significant role in life strategies for elephants. Our research focuses on chemical identification of pheromones functioning during reproduction. We are studying inter-sexual temporal gland (related to the unique male condition known as musth) and urine signals. We have identified (Z)-7-dodecen-1-yl acetate in female preovulatory urine and demonstrated bioactivity of its synthetic form. This compound is also bioactive in many Lepidoptera, making it a good example of convergent evolution of structure and function. Our new directions include sophisticated assessments of behavior resulting from this and other chemical signals, and molecular biological studies to elucidate pheromonal tissue sources and the nature of specific carrier proteins, allowing the sequence from peromone to signal transduction in the neuroreceptive cells of the vomeronasal organ.

Male musth signals may have a role in mate choice by female elephants. These include urinary and temporal gland emissions during musth. We are characterizing the moderate and light volatiles emanating from the temporal gland. Ongoing studies include chemical identification of specific chemical communicators, correlation of these chemicals with testosterone levels in males, and delineation of their behavioral components. Such signals may affect males and females, and the effects may partially depend on the hormonal status of an individual elephant.

Inter-hormonal and inter-pheromonal relationships during reproduction are incompletely understood; few specific signals have been identified in vertebrates. Using elasmobranchs as models, this aspect of our research has focused first on characterizing the hormones operational during reproduction in placental sharks. Our studies have demonstrated some unusual hormone attributes. Now we are characterizing whether reproductive pheromones in elasmobranchs are hormonal blends as in teleosts, or whether they are a different group of chemical compounds. Rasmussen

Radical Copper Oxidases

Radical copper oxidases are a new class of redox metalloenzymes (including the fungal enzymes galactose oxidase and glyoxal oxidase) containing a protein free radical directly coordinated to a copper center. This free radical-coupled Cu complex catalyzes the two-electron oxidation of simple alcohols and aldehydes and the reduction of O2 to hydrogen peroxide, fueling extracellular peroxidases involved in lignin degradation. In these proteins, the free radical is localized on a tyrosine residue covalently crosslinked to a cysteinyl side chain (a Tyr-Cys dimer). The catalytically active enzyme is an intense green color, the result of unusual optical spectra arising from electronic transitions within the copper radical complex. Low energy transitions in the near IR result from interligand redox in this metal complex, ligand-to-ligand charge transfer (LLCT) processes that are closely related to the electron transfer coordinate for substrate oxidation. The active site metal complex is surprisingly flexible, twisting through a pseudorotation distortion when exogenous ligands bind, thereby modulating the basicity of a second tyrosine ligand that serves as a general base in catalysis. Many of these aspects of electronic structure and dynamics of the radical copper oxidases are the focus of active research. Whittaker

Manganese Metalloenzymes

Manganese is an essential element for life, forming the active site for a large number of metalloenzymes catalyzing hydrolytic or redox reactions, including the photosynthetic oxygen evolving complex. We are interested in the Mn redox sites in Mn superoxide dismutase (MnSD, mononuclear Mn) and Mn catalase (MnC, dinuclear Mn), enzymes that provide protection from toxic oxygen metabolites. The key question is: How do interactions between the protein, metal ion and exogenous ligands tune the redox potential and chemistry of these complexes? We are combining the powerful tools of molecular biology with advanced spectroscopic and computational approaches to explore the structure and dynamics of Mn active sites. For MnSD, we find an unexpected temperature dependence for the structures of anion complexes, which change coordination as the temperature is raised. This thermal transition implies that the stability of the active site structure is determined by dynamical features of the complex and that dynamical excitation may play an important role in controlling the energetics of ligand binding and redox. A wide range of projects relating to the chemistry and biology of Mn are in progress. Whittaker

Electronic Spectroscopy of Biological Metal Complexes

Electronic spectroscopy extends structural studies of biomolecules beyond the atomic resolution of X-ray crystallography to a level of structural detail that directly relates to chemistry. The techniques used in these studies span five decades of the electromagnetic spectrum, from microwaves to the ultraviolet and beyond. At the lowest energy, electron paramagnetic resonance (EPR) spectroscopy gives information on the electronic ground state, defining the molecular orbital that contains the unpaired electron in a paramagnetic complex. At higher energy, UV-visible absorption spectroscopy excites orbital transitions between electronic states, giving information on characteristic metal-ligand interaction energies that can be understood in terms of a ligand field or molecular orbital analysis. Polarization spectroscopy (linear dichroism, circular dichroism, and magnetic circular dichroism) can give more detailed information on ground and excited state electronic wave functions using geometric features of light to probe the active site. These experimental approaches can be complemented by spectroscopic modeling and computational biology methods to provide a detailed description of a metalloprotein complex and its interactions. Whittaker

Vibrational Spectroscopy of Metalloprotein Active Sites

Many spectroscopic methods are available for the investigation of structural and functional properties of metal ions in enzymes and proteins. We use electronic, vibrational (especially resonance Raman), and EPR spectroscopy to characterize metal-ion active sites. Our laboratory has a sensitive, state-of-the-art Raman instrument: a fast spectrograph with a liquid N2-cooled CCD detector. We also use a combined FT-IR/FT-Raman instrument for protein and model compound studies.

Our research focuses on the description of the molecular and electronic structures of heme (iron porphyrin), nonheme-iron, and copper enzymes to gain an understanding of the role of the metal ion in enzymatic catalysis. Of particular interest is the biochemistry of O2. Metalloproteins are involved in O2 binding (hemoglobin or hemocyanin) and in oxidative chemistry whereby O2 is reduced and substrates are oxygenated or oxidized. Trapped reaction intermediates and model compounds help us to unravel these complex processes and to define reaction mechanisms. In all projects, modern molecular biology techniques provide site-directed mutants that permit alterations in structures and reactivities. Loehr

Heme Oxygenase

Heme oxygenase is a fascinating system that uses the O2-binding affinity of its heme substrate in the cellular degradation of heme to open-chain biliverdin. These studies are carried out with Paul R. Ortiz de Montellano's group at U.C. San Francisco. The resting heme-heme oxygenase enzyme substrate complex is much like myoglobin: The heme is linked to the enzyme by an iron-histidine bond and the iron exists mainly in a six-coordinate, high-spin state with an additional water ligand. The Fe-NHis bond was identified from its resonance Raman vibration at 216 cm-1 in the Fe(II)-heme complex. The absence of this fingerprint frequency in the H25A mutant clearly identified His25 as the axial ligand. Remarkably, when imidazole was added to the inactive H25A preparation, activity was fully restored. Our current efforts, in collaboration with Angela Wilks at U. of Maryland, examine the structure and activity of several bacterial heme oxygenases. Loehr and Moenne-Loccoz

Oxygen Intermediates of Dinuclear Iron Enzymes

Oxygen Activation by Iron Proteins

Several diiron enzymes react with molecular oxygen to form powerful oxidizing agents important in biology. Examples include (i) ribonucleotide reductase protein R2, which oxidizes its tyrosine 122 to its catalytically important neutral radical form; (ii) methane monooxygenase, whose hydroxylase component oxidizes hydrocarbons to alcohols; (iii) plant desaturases, which oxidize fatty acids to olefins, e.g., stearoyl to oleoyl; and (iv) ferroxidase reactions, in which Fe2+ is oxidized to Fe3+. A common feature of these enzymes appears to be the formation of an initial peroxo intermediate from the reduced enzyme. However, in the respiratory protein, hemerythrin, binding of dioxygen is accomplished by reduction to peroxide in a reaction that is readily reversible. In ribonucleotide reductase, peroxide is similarly formed but decomposes irreversibly to a ferryl intermediate that is capable of carrying out oxidative chemistry. This dichotomy of behavior is reminiscent of the respiratory vs. peroxidase functions of different heme-containing proteins. We are interested in determining common principles that influence the pathways of oxygen utilization. This problem is being approached by structural elucidation of the iron sites in the proteins themselves and in model complexes, as well as by studying mechanisms of their reactions with oxygen-containing substrates. Loehr and Moenne-Loccoz

Regulation of Long Chain Fatty Acid Transport and Oxidation in Mammalian Heart and Liver

The rate-limiting step in ß-oxidation is the conversion of long-chain acyl-CoA to acylcarnitine, a reaction catalyzed by the outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) and inhibited by malonyl-CoA. The acylcarnitine is then translocated across the inner mitochondrial membrane by the carnitine/acylcarnitine translocase and converted back to acyl-CoA by CPTII. This reaction in intact mitochondria is inhibited by malonyl-CoA, the first intermediate in fatty acid synthesis, suggesting coordinated regulation of fatty acid oxidation and synthesis. Although CPTII has been examined in detail, studies on CPTI have been hampered by an inability to purify CPTI in an active form from CPTII. In particular, it has not been conclusively demonstrated that CPTI is even catalytically active, or whether sensitivity of CPTI to malonyl-CoA is an intrinsic property of the enzyme or is contained in a separate regulatory subunit that interacts with CPTI. To address these questions, the genes for human heart muscle M-CPTI and rat liver L-CPTI and CPTII were separately expressed in Pichia pastoris, a yeast with no endogenous CPT activity. High levels of CPT activity were present in purified mitochondrial preparations from both CPTI- and CPTII-expressing strains. Furthermore, CPTI activity was highly sensitive to inhibition by malonyl-CoA while CPTII was not. Thus, CPT catalytic activity and malonyl-CoA sensitivity are contained within a single CPTI-polypeptide in mammalian mitochondrial membranes. My laboratory is the first to describe the kinetic characteristics for the yeast-expressed CPTIs, the first such report for a CPTI enzyme in the absence of CPTII. Both yeast-expressed M-CPTI and L-CPTI are inactivated by detergent solubilization. However, removal of the detergent in the presence of phospholipids resulted in the recovery of malonyl-CoA-sensitive CPTI activity, suggesting that CPTI requires a membranous environment. CPTI is thus reversibly inactivated by detergents. We have isolated and sequenced the promoter region of the gene for the human heart M-CPTI. We are mapping the malonyl-CoA and substrate binding sites in human heart M-CPTI and liver L-CPTI by site-directed mutagenesis and chemical modification studies using residue-specific reagents. We will determine the structural basis for the high malonyl-CoA sensitivity of M-CPTI by constructing chimeras between M-CPTI and L-CPTI and by site-directed mutagenesis. We will prepare the expressed highly purified human heart M-CPTI and liver L-CPTI for structural characterization studies. Finally, we plan to study the regulation of human heart M-CPTI expression by hormonal, developmental and dietary factors. Our goal is to elucidate the molecular mechanism of the regulation of fatty acid transport and oxidation in mammalian cells. Woldegiorgis