体重和盐度对中国蛤蜊耗氧率和排氨率的影响

采用室内实验生态方法研究了不同软体部干重((1.022 ±0.821)、(0.557±0.266)、(0.303±0.277) g)和盐度(13、18、23、28、33)对中国蛤蜊(Mactra chinensis Philippi)耗氧率和排氨率的影响。结果表明:盐度、个体大小对中国蛤蜊耗氧率的影响极显著(P＜0.01),二者的交互作用对中国蛤蜊耗氧率影响显著(P＜0.05);中国蛤蜊单位体重耗氧率(R₀)与软体部干重(W)负相关,符合幂函数方程R₀=aW^-b, 其中a值的取值范围是0.695-1.762,平均值为1.449,b值的取值范围是0.446-0.587,平均值为0.542。盐度、个体大小对中国蛤蜊排氨率影响也极显著(P＜0.01);随着中国蛤蜊个体的增大,其单位体重排氨率逐渐降低;排氨率与其软体部干重呈负相关,它们之间可以用幂函数R_N=a₀W^-b₀表示。单位体重耗氧率和排氨率与盐度(S)、软体部干重(W)的二元线性回归方程分别为: R_O=2.111-1.817W+0.49S (R²=0.546, F=34.294, P＜0.001);R_N=168.186-120.589W+1.734S (R²=0.561, F=36.418, P＜0.001)。     

我国东北土壤有机碳、无机碳含量与土壤理化性质的相关性

根据黑龙江、吉林、辽宁省和内蒙古地区相关历史资料数据,分析了我国东北表层土壤(0-50 cm)土壤相关理化性质与有机碳、无机碳的相关性,得到如下结论:土壤全氮、碱解氮、全磷、速效磷、速效钾、K⁺离子交换量、Fe₂O₃、P₂O₅、总孔隙度均与土壤有机碳含量呈显著正相关(R²=0.10-0.94, n=38-345, P<0.0001),但与土壤无机碳含量则大多呈显著负相关(R²=0.11-0.30, n=37-122, P<0.01);与此相反,土壤pH值、容重与土壤有机碳呈负相关(R²=0.36-0.42,n=41-304, P<0.0001),而与无机碳呈显著正相关(R²=0.29-0.31,n=39-125, P <0.01)。表层土壤有机碳、无机碳与土壤理化性质呈相反变化趋势的结果说明,由于土壤利用方式变化所导致的土壤理化性质改变对土壤无机碳和有机碳可能具有相反影响。在研究土壤碳平衡过程中,应该充分考虑这种关系所导致的相互补偿作用,即有机碳的增加,可能意味着无机碳的减少,或者反之。目前研究中普遍忽略无机碳的变化,可能导致生态系统碳收支计算显著偏差,所获得的经验拟合方程有利于对我国东北地区土壤碳平衡研究产生的这种偏差进行粗略估计。     

黄土高原刺槐人工林地表凋落物对土壤呼吸的贡献

于黄土高原沟壑区王东沟小流域26年刺槐人工林(Robinia pseudoacacia)中,设置对照(CK)、去除凋落物(no litter, NL)和倍增凋落物(double litter, DL)3个处理,利用Li-8100系统测定各处理的土壤呼吸速率。结果表明,添加或去除凋落物显著影响土壤呼吸(P = 0.091-0.099),与对照(CK)的土壤呼吸速率(3.23 μmol m^-2 s^-1)相比,添加凋落物(DL)使土壤呼吸速率增加26%,去除凋落物(NL)使土壤呼吸速率减少22%。NL、CK和DL的累积土壤呼吸分别为631、787和973 g C m^-2a^-1。各处理土壤呼吸速率与土壤温度呈显著的指数关系(R²=0.81-0.90,P < 0.0001),但与土壤水分的关系不明显。NL、CK和DL的Q₁₀依次为1.92、2.29和2.31。地表凋落物对土壤呼吸年平均贡献量为20%。相关性分析表明,各测定日地表凋落物贡献与土壤温度(r=0.54,P < 0.05)或土壤水分关系显著(r=0.68, P < 0.05)。刺槐人工林地表凋落物的输入量为213 g C m^-2a^-1,大于凋落物引起的呼吸量156 g C m^-2a^-1。在黄土区通过植被恢复治理水土流失过程中,随着地表凋落物的积累,林地生态系统的碳汇功能将逐步得到加强。     

氧化磷酸化抑制剂对光滑球拟酵母糖酵解速度的影响

研究了不同浓度电子传递链抑制剂 ( 鱼藤酮和抗霉素 A) 和 F_OF₁-ATPase 抑制剂 ( 寡霉素 ) 对光滑球拟酵母胞内 ATP 水平、葡萄糖消耗速度、糖酵解途径关键酶的影响 . 在培养液中添加 10 mg/L 鱼藤酮和抗霉素 A ,相对于对照组,胞内 ATP 分别下降了 43% 和 27.7% ,使糖酵解关键酶磷酸果糖激酶 (PFK) 的活性分别提高 340% 和 230% ,从而导致葡萄糖消耗速度增加 360% 和 240% ,丙酮酸生成速度提高了 17% 和 8.5%. 改变胞内 ATP 水平并不影响糖酵解途径其他关键酶 HK 、 PK 活性 . 微量的寡霉素 (0.05 mg/L) 可使胞内 ATP 含量下降 64.3% ,当培养液中寡霉素浓度达到 0.4 mg/L 时,细胞不能继续生长,葡萄糖消耗速度和丙酮酸的生成速度却随着寡霉素浓度 ( 小于 0.6 mg/L) 的增加而增加 . 表明氧化磷酸化途径中, ATPase 决定着 ATP 的生成 . 降低胞内 ATP 含量能显著提高 PFK 活性 (r²=0.9971) ,葡萄糖消耗速度 (r²= 0.9967) 以及丙酮酸生产速度 (r²= 0.965) ,葡萄糖消耗速度的增加是糖酵解途径中关键酶 PFK 活性 (r² = 0.9958) 和 PK 活性 (r²= 0.8706) 增加所导致的 . 这一结果有利于揭示真核微生物细胞中氧化磷酸化与中心代谢途径 ( 酵解 ) 的关系 .     

Gc、Pi蛋白亚型的快速微量电泳分析

用快速微量等电聚焦技术对190名北京地区汉族健康人血清Gc蛋白亚型、Pi蛋白亚型进行分型鉴定和基因频率调查.上样量为1.5μl,电泳和染色各0.5h.Gc^1F=0.4891,Gc^1S=0.2432,Gc²=0.2678.观察值与期望值吻合良好.(∑X²=1.404,0.7<P<0.8).Pi^M₁=0.7542,Pi^M₂=0.1808,Pi^M₃=0.0650,观察值与期望值吻合也良好,(∑X²=1.1233,0.7<P<0.8).     

鄱阳湖苔草湿地甲烷释放特征

2009年5月-2010年4月在鄱阳湖南矶湿地国家级自然保护区选择以灰化苔草为建群种的洲滩,设置土壤-植物系统(TC)、剪除植物地上部分 (TJ)2个试验处理,利用密闭箱-气相色谱法测定了鄱阳湖典型苔草湿地的甲烷(CH₄)释放通量。结果表明:1)TC、TJ 2个试验处理CH₄释放速率变化范围分别为-0.094-17.75 mg · m^-2 · h^-1、-0.122-19.16 mg · m^-2 · h^-1,均表现出明显的季节变化规律;2)地表未淹水期间,剪草处理CH₄释放显著高于非剪草处理(t=2.69, P<0.05);地表淹水达到15 cm后,剪草处理CH₄释放明显低于非剪草处理。3)土壤5 cm温度、土壤水分与2处理非淹水期间CH₄释放速率均呈显著正相关,是非淹水期间CH₄通量变化的主要控制因子,2因子能够共同解释非淹水期苔草湿地65%-74%的CH₄通量变异;4)试验期间,苔草湿地CH₄释放量约为12.77 gC/m²,相当于同期土壤有机质分解碳排放量的4%,甲烷释放的碳消耗不足苔草湿地年NPP的1%。     

抗人IL-6单克隆抗体的制备及鉴定

用基因重组人IL-6免疫Balb/c小鼠,采用小鼠杂交瘤技术,筛选克隆到分泌抗人重组IL-6单克隆抗体的杂交瘤细胞株,并对其中2H₂、 1D₂ 和4B₄瘤细胞株进行了鉴定.其抗体类别均为IgG,亚类分别为IgG1和IgG2a.用多种细胞因子和无关蛋白的鉴别试验结果证实它们均特异地识别rhIL-6.免疫转染结果显示,该单抗识别分子质量为21 ku的IL-6单一条带.IL-6单克隆抗体的亲和常数K_aff= 1.62×10⁹ (mol/L)^-1.     

Partitioning soil respiration of temperate forest ecosystems in northeastern China

Quantifying soil respiration components and their relations to environmental controls are essential to estimate both local and regional carbon (C) budgets of forest ecosystems. In this study, we used the trenching-plot and infrared gas exchange analyzer approaches to determine heterotrophic (R_H) and autotrophic respiration (R_A) in the soil surface CO₂ flux for six major temperate forest ecosystems in northeastern China. The ecosystems were: Mongolian oak forest (dominated by Quercus mongolica), aspen-birch forest (dominated by Populous davidiana and Betula platyphylla), mixed wood forest (composed of P. davidiana, B. platyphylla, Fraxinus mandshurica, Tilia amurensis, Acer amono, etc.), hardwood forest (dominated by F. mandshurica, Juglans mandshurica, and Phellodendron amurense), Korean pine (Pinus koraiensis), and Dahurian larch (Larix gmelinii) plantations, representing the typical secondary forest ecosystems in this region. Our specific objectives were to: (1) quantify R_H and its relationship with the environmental factors of the forest ecosystems, (2) characterize seasonal dynamics in the contribution of root respiration to total soil surface CO₂ flux (RC), and (3) compare annual CO₂ fluxes from R_H and R_A among the six forest ecosystems. Soil temperature, water content, and their interactions significantly affected R_H in the ecosystems and accounted for 46.5%–78.8% variations in R_H. However, the environmental controlling factors of R_H varied with ecosystem types: soil temperature in hardwood and Dahurian larch forest ecosystems, soil temperature, and water content in the others. The RC for hardwood, poplar-birch, mixed wood, Mongolian oak, Korean pine, and Dahurian larch forest ecosystems varied between 32.40%–51.44%, 39.72%–46.65%, 17.94%–47.74%, 34.31%–37.36%, 33.78%–37.02%, and 14.39%–35.75%, respectively. The annual CO₂ fluxes from R_H were significantly greater than those from R_A for all the ecosystems, ranging from 337–540 g Cm^-2a^-1 and 88‐331 gCm^-2a^-1 for R_H and R_A, respectively. The annual CO₂ fluxes from R_H and R_A differed significantly among the six forest ecosystems.     

华北平原玉米田生态系统光合作用特征及影响因素

采用涡度相关法对华北平原夏玉米田进行了连续4a(2003-2006年)的碳通量观测,结果表明:夏玉米田生态系统初始量子效率(α)、最大光合速率(P_max)、暗呼吸速率(R_d)和总初级生产力(GPP)随作物生长发育而变化。在夏玉米生育前期和后期,α、P_max、R_d和GPP都比较小,其最大值出现在抽穗期/灌浆期。2003-2006年,夏玉米生长季平均α、P_max、R_d的范围分别为0.054-0.124 μmol/μmol、1.72-2.93 mg CO₂ · m^-2 · s^-1、0.23-0.38 mg CO₂ · m^-2 · s^-1。α、P_max和R_d均随叶面积指数(LAI)增加呈指数增长。2003-2006年夏玉米生长季GPP总量分别为806.2、741.5、703.0、817.4 g C/m²,年际差异较大。玉米田生态系统GPP随温度升高呈指数增长。在玉米营养生长阶段,GPP随LAI增加而增大,两者之间的关系可用直角双曲线方程来表示;生殖生长阶段,GPP随LAI降低而下降.相同LAI下,生殖生长阶段的GPP明显低于营养生长阶段。     

苹果三维树冠的净光合速率分布模拟

构建三维树冠光合模型可模拟出叶片净光合速率(P_n)、气孔导度(G_s)和光能利用效率(LUE)在树冠内的三维分布。以17年生纺锤形"富士"苹果树(Malus domestica Borkh. cv. ‘Fuji’)为试材,通过实测确定三维树冠内叶片和辐射分布,根据不同部位叶片最大光合速率经验公式模拟叶片P_n 在三维树冠空间内分布,并据2007-2009年测定数据拟合相关模型参数。模拟表明,苹果树冠叶片P_n 和辐射的三维分布相似,在树冠上部P_n 三维分布比较平缓,然后随辐射的减少而迅速降低。高辐射条件下(PAR=1500 μmol·m^-2·s^-1),从树冠上部3 m处降到到1 m,平均相对辐射从71.18%降到8.05%,减少了89%,叶片平均P_n从15.05 μmol·m^-2·s^-1降到1.92 μmol·m^-2·s^-1,减少了87%。单位体积小室内的总净光合速率大小主要取决于叶面积密度,部分取决于P_n。G_s三维分布与P_n相似,而LUE分布与辐射相反,中下部高,上部低。根据光合机理模型、树冠内辐射和叶面积三维分布可模拟出苹果三维树冠内叶片的P_n、G_s和LUE分布,该模型参数少,可方便用于其它果树三维光合模型构建和果树整形修剪研究。     

Refined crystal structures of human Ca(2+)/Zn(2+)-binding S100A3 protein characterized by two disulfide bridges

S100A3, a member of the EF-hand-type Ca²⁺-binding S100 protein family, is unique in its exceptionally high cysteine content and Zn²⁺ affinity. We produced human S100A3 protein and its mutants in insect cells using a baculovirus expression system. The purified wild-type S100A3 and the pseudo-citrullinated form (R51A) were crystallized with ammonium sulfate in N,N-bis(2-hydroxyethyl)glycine buffer and, specifically for postrefolding treatment, with Ca²⁺/Zn²⁺ supplementation. We identified two previously undocumented disulfide bridges in the crystal structure of properly folded S100A3: one disulfide bridge is between Cys30 in the N-terminal pseudo-EF-hand and Cys68 in the C-terminal EF-hand (SS1), and another disulfide bridge attaches Cys99 in the C-terminal coil structure to Cys81 in helix IV (SS2). Mutational disruption of SS1 (C30A + C68A) abolished the Ca²⁺ binding property of S100A3 and retarded the citrullination of Arg51 by peptidylarginine deiminase type III (PAD3), while SS2 disruption inversely increased both Ca²⁺ affinity and PAD3 reactivity in vitro. Similar backbone structures of wild type, R51A, and C30A + C68A indicated that neither Arg51 conversion by PAD3 nor SS1 alters the overall dimer conformation. Comparative inspection of atomic coordinates refined to 2.15−1.40 Å resolution shows that SS1 renders the C-terminal classical Ca²⁺-binding loop flexible, which are essential for its Ca²⁺ binding properties, whereas SS2 structurally shelters Arg51 in the metal-free form. We propose a model of the tetrahedral coordination of a Zn²⁺ by (Cys)₃His residues that is compatible with SS2 formation in S100A3.     

Crystal Structure of Sulfide:Quinone Oxidoreductase from Acidithiobacillus ferrooxidans: Insights into Sulfidotrophic Respiration and Detoxification

Sulfide:quinone oxidoreductase from the acidophilic and chemolithotrophic bacterium Acidithiobacillus ferrooxidans was expressed in Escherichia coli and crystallized, and its X-ray molecular structure was determined to 2.3 Å resolution for native unbound protein in space group P4₂2₁2 . The decylubiquinone-bound structure and the Cys160Ala variant structure were subsequently determined to 2.3 Å and 2.05 Å resolutions, respectively, in space group P6₂22  . The enzymatic reaction catalyzed by sulfide:quinone oxidoreductase includes the oxidation of sulfide compounds H₂S, HS⁻, and S²⁻ to soluble polysulfide chains or to elemental sulfur in the form of octasulfur rings; these oxidations are coupled to the reduction of ubiquinone or menaquinone. The enzyme comprises two tandem Rossmann fold domains and a flexible C-terminal domain encompassing two amphipathic helices that are thought to provide for membrane anchoring. The second amphipathic helix unwinds and changes its orientation in the hexagonal crystal form. The protein forms a dimer that could be inserted into the membrane to a depth of approximately 20 Å. It has an endogenous flavin adenine dinucleotide (FAD) cofactor that is noncovalently bound in the N-terminal domain. Several wide channels connect the FAD cofactor to the exterior of the protein molecule; some of the channels would provide access to the membrane. The ubiquinone molecule is bound in one of these channels; its benzoquinone ring is stacked between the aromatic rings of two conserved Phe residues, and it closely approaches the isoalloxazine moiety of the FAD cofactor. Two active-site cysteine residues situated on the re side of the FAD cofactor form a branched polysulfide bridge. Cys356 disulfide acts as a nucleophile that attacks the C4A atom of the FAD cofactor in electron transfer reaction. The third essential cysteine Cys128 is not modified in these structures; its role is likely confined to the release of the polysulfur product.     

Solubilization and purification of cytochrome a1 from Nitrosomonas

Cytochrome a₁ was solubilized with Triton X-100 from a membrane-envelope preparation of Nitrosomonas and partially purified by repeated fractionation with (NH₄)₂SO₄. The purified fraction of cytochrome a₁ was enriched over the crude extract by a factor of 16 and 300 with respect to protein and c-type cytochrome, respectively. The cytochrome was characterized as cytochrome a₁ on the basis of (a) reduced absorption maxima at 444 nm and 595 nm, (b) acid acetone extractibility and ether solubility of the heme and (c) absorption maximum of 587 nm of the ferro-hemochrome in alkaline pyridine. The α absorption band shifted from 600 nm to 595 nm upon solubilization of the cytochrome with Triton X-100. Spectral shifts were observed in the presence of cyanide and azide and the cytochrome changed with aging to a form with a reduced absorption band at 422 nm. Cytochrome a₁ was reduced anaerobically in the presence of reduced mammalian cytochrome c and was rapidly reoxidized in the presence of O₂. CO caused a shift in the soret peak of the reduced form but did not prevent reoxidation of cytochrome a₁ in the presence of CO-O₂ (95:5, v/v).     

EmrE dimerization depends on membrane environment

The small multi-drug resistant (SMR) transporter EmrE functions as a homodimer. Although the small size of EmrE would seem to make it an ideal model system, it can also make it challenging to work with. As a result, a great deal of controversy has surrounded even such basic questions as the oligomeric state. Here we show that the purified protein is a homodimer in isotropic bicelles with a monomer–dimer equilibrium constant (K_MD^2D) of 0.002–0.009 mol% for both the substrate-free and substrate-bound states. Thus, the dimer is stabilized in bicelles relative to detergent micelles where the K_MD^2D is only 0.8–0.95 mol% (Butler et al. 2004). In dilauroylphosphatidylcholine (DLPC) liposomes K_MD^2D is 0.0005–0.0008 mol% based on Förster resonance energy transfer (FRET) measurements, slightly tighter than bicelles. These results emphasize the importance of the lipid membrane in influencing dimer affinity.     

Structural and Functional Model for Ionic (K/Na) and pH Dependence of GTPase Activity and Polymerization of FtsZ, the Prokaryotic Ortholog of Tubulin

Bacterial cell division occurs through the formation of a protein ring (division ring) at the site of division, with FtsZ being its main component in most bacteria. FtsZ is the prokaryotic ortholog of eukaryotic tubulin; it shares GTPase activity properties and the ability to polymerize in vitro. To study the mechanism of action of FtsZ, we used molecular dynamics simulations of the behavior of the FtsZ dimer in the presence of GTP-Mg²⁺ and monovalent cations. The presence of a K⁺ ion at the GTP binding site allows the positioning of one water molecule that interacts with catalytic residues Asp235 and Asp238, which are also involved in the coordination sphere of K⁺. This arrangement might favor dimer stability and GTP hydrolysis. Contrary to this, Na⁺ destabilizes the dimer and does not allow the positioning of the catalytic water molecule. Protonation of the GTP gamma-phosphate, simulating low pH, excludes both monovalent cations and the catalytic water molecule from the GTP binding site and stabilizes the dimer. These molecular dynamics predictions were contrasted experimentally by analyzing the GTPase and polymerization activities of purified Methanococcus jannaschii and Escherichia coli FtsZ proteins in vitro.     

Fructose 1-phosphate is the preferred effector of the metabolic regulator Cra of Pseudomonas putida

The catabolite repressor/activator (Cra) protein is a global sensor and regulator of carbon fluxes through the central metabolic pathways of Gram-negative bacteria. To examine the nature of the effector (or effectors) that signal such fluxes to the protein of Pseudomonas putida, the Cra factor of this soil microorganism has been purified and characterized and its three-dimensional structure determined. Analytical ultracentrifugation, gel filtration, and mobility shift assays showed that the effector-free Cra is a dimer that binds an operator DNA sequence in the promoter region of the fruBKA cluster. Furthermore, fructose 1-phosphate (F1P) was found to most efficiently dissociate the Cra-DNA complex. Thermodynamic parameters of the F1P-Cra-DNA interaction calculated by isothermal titration calorimetry revealed that the factor associates tightly to the DNA sequence 5′-TTAAACGTTTCA-3′ (K_D = 26.3 ± 3.1 nm) and that F1P binds the protein with an apparent stoichiometry of 1.06 ± 0.06 molecules per Cra monomer and a K_D of 209 ± 20 nm. Other possible effectors, like fructose 1,6-bisphosphate, did not display a significant affinity for the regulator under the assay conditions. Moreover, the structure of Cra and its co-crystal with F1P at a 2-Å resolution revealed that F1P fits optimally the geometry of the effector pocket. Our results thus single out F1P as the preferred metabolic effector of the Cra protein of P. putida.     

Function of two β-carotenes near the D1 and D2 proteins in photosystem II dimers

The antenna proteins in photosystem II (PSII) not only promote energy transfer to the photosynthetic reaction center (RC) but provide also an efficient cation sink to re-reduce chlorophyll a if the electron transfer (ET) from the Mn-cluster is inhibited. Using the newest PSII dimer crystal structure (3.0 Å resolution), in which 11 β-carotene molecules (Car) and 14 lipids are visible in the PSII monomer, we calculated the redox potentials (E_m) of one-electron oxidation for all Car (E_m(Car)) by solving the Poisson-Boltzmann equation. In each PSII monomer, the D1 protein harbors a previously unlocated Car (Car_D1) in van der Waals contact with the chlorin ring of Chl_Z(D1). Each Car_D1 in the PSII dimer complex is located in the interface between the D1 and CP47 subunits, together with another four Car of the other PSII monomer and several lipid molecules. The proximity of Car bridging between Car_D1 and plastoquinone/Q_A may imply a direct charge recombination of Car⁺Q_A⁻. The calculated E_m(Car_D1) and E_m(Chl_Z(D1)) are, respectively, 83 and 126 mV higher than E_m(Car_D2) and E_m(Chl_Z(D2)), which could explain why Car_D2⁺ and Chl_Z(D2)⁺ are observed rather than the corresponding Car_D1⁺ and Chl_Z(D1)⁺.     

Kinetic and thermodynamic properties of the folding and assembly of formate dehydrogenase

The folding mechanism and stability of dimeric formate dehydrogenase from Candida methylica was analysed by exposure to denaturing agents and to heat. Equilibrium denaturation data yielded a dissociation constant of about 10⁻¹³ M for assembly of the protein from unfolded chains and the kinetics of refolding and unfolding revealed that the overall process comprises two steps. In the first step a marginally stable folded monomeric state is formed at a rate (k₁) of about 2 × 10⁻³ s⁻¹ (by deduction k₋₁ is about10⁻⁴ s⁻¹) and assembles into the active dimeric state with a bimolecular rate constant (k₂) of about 2 × 10⁴ M⁻¹ s⁻¹. The rate of dissociation of the dimeric state in physiological conditions is extremely slow (k₋₂ ∼ 3 × 10⁻⁷ s⁻¹).     

A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k_cat/K_m = 1000 ± 100 m⁻¹ s⁻¹ versus 700 ± 100 m⁻¹ s⁻¹). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k_cat/K_m = 80 ± 40 m⁻¹ s⁻¹). In the presence of 3-HSA the K_mapp for O₂ was 100 ± 10 μm. The crystal structure of HsaA to 2.5-Å resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme''s substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val³⁶⁷–Val³⁹⁴) could adopt two conformations differing by a rigid body rotation of 25° around Arg³⁶⁶. This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme''s substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids.