We recently reported the detection of methanol emissions from leaves (R. MacDonald, R. Fall 119931 Atmos Environ 27A: 1709-171 3). This could represent a substantial flux of methanol to the atmosphere. Leaf methanol production and emission have not been investigated in detail, in part because of difficulties in sampling and analyzing methanol. In this study we used an enzymatic method to convert methanol to a fluorescent product and verified that leaves from severa1 species emit methanol. Methanol was emitted almost exclusively from the abaxial surfaces of hypostomatous leaves but from both surfaces of amphistomatous leaves, suggesting that methano1 exits leaves via stomates. The role of stomatal conductance was verified in experiments in which stomates were induced to close, resulting in reduced methanol. Free methanol was detected in bean leaf extracts, ranging from 26.8 pg g-' fresh weight in young leaves to 10.0 pg g-' fresh weight in older leaves. Methanol emission was related to leaf development, generally declining with increasing leaf age after leaf expansion; this is consistent with volatilization from a cellular pool that declines in older leaves. It is possible that leaf emission could be a major source of methanol found in the atmosphere of forests.

One of the best-studied examples of a class A beta-lactamase is Escherichia coli TEM-1 beta-lactamase. In this class of enzymes, the active-site serine residue takes on the role of a nucleophile and carries out beta-lactam hydrolysis. Here, the structures of the wild-type and the S70G enzyme determined to 1.55 and 2.1 A, respectively, are presented. In contrast to the previously reported 1.8 A structure, the active site of the wild-type enzyme (1.55 A) structure does not contain sulfate and Ser70 appears to be in the deprotonated form. The X-ray crystal structure of the S70G mutant has an altered Ser130 side-chain conformation that influences the positions of water molecules in the active site. This change allows an additional water molecule to be positioned similarly to the serine hydroxyl in the wild-type enzyme. The structure of the mutant enzyme suggests that this water molecule can assume the role of an active-site nucleophile and carry out noncovalent catalysis. The drop in activity in the mutant enzyme is comparable to the drop observed in an analogous mutation of the nucleophilic serine in alkaline phosphatase, suggesting common chemical principles in the utilization of nucleophilic serine in the active site of different enzymes.

The hyperthermophilic bacterium Thermotoga maritima encodes a gene sharing sequence similarities with several known genes for alkaline phosphatase (AP). The putative gene was isolated and the corresponding protein expressed in Escherichia coli, with and without a predicted signal sequence. The recombinant protein showed phosphatase activity toward the substrate p-nitrophenyl-phosphate with a k(cat) of 16 s(-1) and a K(m) of 175 microM at a pH optimum of 8.0 when assayed at 25 degrees C. T. maritima phosphatase activity increased at high temperatures, reaching a maximum k(cat) of 100 s(-1), with a K(m) of 93 microM at 65 degrees C. Activity was stable at 65 degrees C for >24 h and at 90 degrees C for 5 h. Phosphatase activity was dependent on divalent metal ions, specifically Co(II) and Mg(II). Circular dichroism spectra showed that the enzyme gains secondary structure on addition of these metals. Zinc, the most common divalent metal ion required for activity in known APs, was shown to inhibit the T. maritima phosphatase enzyme at concentrations above 0.3 moles Zn: 1 mole monomer. All activity was abolished in the presence of 0.1 mM EDTA. The T. maritima AP primary sequence is 28% identical when compared with E. coli AP. Based on a structural model, the active sites are superimposable except for two residues near the E. coli AP Mg binding site, D153 and K328 (E. coli numbering) corresponding to histidine and tryptophan in T. maritima AP, respectively. Sucrose-density gradient sedimentation experiments showed that the protein exists in several quaternary forms predominated by an octamer.

Analysis of sequence alignments of alkaline phosphatases revealed a correlation between metal specificity and certain amino acid side chains in the active site that are metal-binding ligands. The Zn 2؉ -requiring Escherichia coli alkaline phosphatase has an Asp at position 153 and a Lys at position 328. Co 2؉ -requiring alkaline phosphatases from Thermotoga maritima and Bacillus subtilis have a His and a Trp at these positions, respectively. The mutations D153H, K328W, and D153H/ K328W were induced in E. coli alkaline phosphatase to determine whether these residues dictate the metal dependence of the enzyme. The wild-type and D153H enzymes showed very little activity in the presence of Co 2؉ , but the K328W and especially the D153H/K328W enzymes effectively use Co 2؉ for catalysis. Isothermal titration calorimetry experiments showed that in all cases except for the D153H/K328W enzyme, a possible conformation change occurs upon binding Co 2؉ . These data together indicate that the active site of the D153H/ K328W enzyme has been altered significantly enough to allow the enzyme to utilize Co 2؉ for catalysis. These studies suggest that the active site residues His and Trp at the E. coli enzyme positions 153 and 328, respectively, at least partially dictate the metal specificity of alkaline phosphatase.

While screening aerobic, heterotrophic marine bacteria for production of volatile organic compounds, we found that a group of isolates produced substantial amounts of acetone. Acetone production was confirmed by gas chromatography, gas chromatography-mass spectrometry, and high-performance liquid chromatography. The major acetone producers were identified as nonclinical Vibrio species. Acetone production was maximal in the stationary phase of growth and was stimulated by addition of L-leucine but not the other common amino acids, suggesting that leucine degradation leads to acetone formation. Acetone production by marine vibrios may contribute to the dissolved organic carbon associated with phytoplankton, and some of the acetone produced may be volatilized to the atmosphere. It is now known that the biosphere emits large quantities of volatile organic compounds (VOCs) into the atmosphere. Some VOCs, like methane, isoprene, and monoterpenes, are emitted from terrestrial sources at levels of hundred of millions of metric tons per year on a global basis and have important effects on atmospheric chemistry and global climate (12, 20). Oceans are significant sources of light hydrocarbons, including ethane, ethylene, propane, and propylene (reviewed in reference 26). Presumably, many of these marine hydrocarbons are produced by phytoplankton, which are known to produce a complex array of VOCs (29). Recently, it was reported that the VOC isoprene is produced in seawater (6, 19, 22). We set out to determine if marine microorganisms could be responsible for this production. During these investigations we detected substantial production of acetone by marine heterotrophic bacteria; our observations concerning this acetone production are reported in this paper. MATERIALS AND METHODS Screening marine bacteria for acetone production. Marine heterotrophic bacteria were isolated from seawater by inoculating seawater onto enriched seawater agar containing 80% (vol/vol) seawater, 0.1% (wt/vol) glucose, 0.1% (wt/vol) Bacto Tryptone, and 0.01% (wt/vol) yeast extract. The plates were incubated at 20 to 22ЊC and, after visible colonies were obtained, were sealed with Parafilm for 4 to 16 h to allow VOCs to accumulate in the headspace above the agar layer. Gas samples were removed with a gas-tight syringe by passing a side port needle though the Parafilm. Usually, 0.25-to 1.0-cm 3 gas samples were removed (approximately 1 to 4% of the available headspace) for analysis by gas chromatography (GC). We used two different instruments and detectors for GC. In our initial experiments we used a Photovac model 10S portable instrument (GC method A); this instrument was equipped with a 6-foot (ca. 183-cm) type CPSil-5 capillary column and a photoionization detector. The carrier gas was hydrocarbon-free air (8 cm 3 min Ϫ1), and the column was typically operated at 23ЊC; under these conditions acetone eluted at 73 Ϯ 2 s. In later experiments, we used a Hewlett-Packard model 5790A instrument equipped with a flame ionization detector and 30-m type DB-...