Supplementary Table 1| Steady-state parameters for cleavage of 5-nitrobenzisoxazole by representative Kemp eliminases a Catalyst k cat (s-1) K M (mM) k cat /K M (M-1 s-1) (k cat /K M)/k AcO-b k cat /k uncat c Ref 34E4 d 0.66 0.12 5.5 x 10 3 9.5 x 10 7 5.7 x 10 5 11

A general approach for the computational design of enzymes to catalyze arbitrary reactions is a goal at the forefront of the field of protein design. Recently, computationally designed enzymes have been produced for three chemical reactions through the synthesis and screening of a large number of variants. Here, we present an iterative approach that has led to the development of the most catalytically efficient computationally designed enzyme for the Kemp elimination to date. Previously established computational techniques were used to generate an initial design, HG-1, which was catalytically inactive. Analysis of HG-1 with molecular dynamics simulations (MD) and X-ray crystallography indicated that the inactivity might be due to bound waters and high flexibility of residues within the active site. This analysis guided changes to our design procedure, moved the design deeper into the interior of the protein, and resulted in an active Kemp eliminase, HG-2. The cocrystal structure of this enzyme with a transition state analog (TSA) revealed that the TSA was bound in the active site, interacted with the intended catalytic base in a catalytically relevant manner, but was flipped relative to the design model. MD analysis of HG-2 led to an additional point mutation, HG-3, that produced a further threefold improvement in activity. This iterative approach to computational enzyme design, including detailed MD and structural analysis of both active and inactive designs, promises a more complete understanding of the underlying principles of enzymatic catalysis and furthers progress toward reliably producing active enzymes.computational protein design | de novo enzyme design | proton transfer T he high efficiency, chemoselectivity, regio-and stereospecificity, and biodegradability of enzymes make them extremely attractive catalysts. However, the finite repertoire of naturally occurring enzymes limits their applicability to broad problems in biotechnology. A general method for the computational design of enzymes that can efficiently catalyze arbitrary chemical reactions would allow the benefits of enzymatic catalysis to be applied to chemical transformations of interest that are currently inaccessible via natural enzymes. Bolon and Mayo provided important early evidence that such an approach is feasible (1), which motivated significant progress toward this goal in recent years. Using quantum mechanics-based active site design and the Rosetta software suite, Baker, Houk, and coworkers designed enzymes for three chemically unrelated nonnatural reactions in a variety of catalytically inert scaffolds (2-4).In early incarnations of computational protein design, a strategy for methods development was put forth in terms of the so-called "protein design cycle" in which experimental evaluation of an initial design is used to inform adjustments to the design process for subsequent rounds of design (5, 6). Ideally, these steps would be continued iteratively until the protein sequences predicted by the algorithm exhibit the desired char...

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Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion-holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by sidechains or bridging water molecules and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (kcat/KM) of 400 M−1s−1 for the cleavage of a p-nitrophenyl ester. Kinetic experiments indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.

Mercury is a ubiquitous pollutant that when absorbed is extremely toxic to a wide variety of biochemical processes. Mercury (II) is a strong, ''invisible'' poison that is rapidly absorbed by tissues of the intestinal tract, kidneys, and liver upon ingestion. In this study, a novel fluorescence-based biosensor is presented that allows for the direct monitoring of the uptake and distribution of the metal under noninvasive in vivo conditions. With the introduction of a cysteine residue at position 205, located in close proximity to the chromophore, the green fluorescent protein (GFP) from Aequorea victoria was converted into a highly specific biosensor for this metal ion. The mutant protein exhibits a dramatic absorbance and fluorescence change upon mercuration at neutral pH. Absorbance and fluorescence properties with respect to the metal concentration exhibit sigmoidal binding behavior with a detection limit in the low nanomolar range. Time-resolved binding studies indicate rapid subsecond binding of the metal to the protein. The crystal structures obtained of mutant eGFP205C indicate a possible access route of the metal into the core of the protein. To our knowledge, this engineered protein is a first example of a biosensor that allows for noninvasive and real-time imaging of mercury uptake in a living cell. A major advantage is that its expression can be genetically controlled in many organisms to enable unprecedented studies of tissue specific mercury uptake.Keywords: mercury; biosensor; GFP; fluorescence Supplemental material: see www.proteinscience.org Understanding the uptake and distribution of toxic metals is crucial to the diagnosis and identification of heavy metal induced diseases and contaminations. Many of these metals are hazardous when absorbed at even minute concentrations, causing severe neurobehavioral effects and cardiovascular and digestive diseases. Mercury, in particular, is a very potent inhibitor to many protein functions as it can readily be absorbed in the lungs, through the skin, or by ingestion. Many of the diseases induced by the absorption of mercuric compounds are most frequently caused by a chronic low-level exposure to the metal or by temporary high-level exposure due to human negligence and pollution. The uptake of mercury through inhalation predominantly affects bronchial tissues, whereas mercury absorption by ingestion primarily affects stomach, intestine, kidneys, and liver. Hydrophobic organomercurials can also cross the blood-brain barrier, Reprint requests to: Martin Sagermann, University of California, Santa Barbara, 1631 Physical Science North, Santa Barbara, CA 93106-9510, USA; e-mail: sagermann@chem.ucsb.edu; fax: (805) 893-4120.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

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