Structures of the Omicron spike trimer with ACE2 and an anti-Omicron antibody.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has become the dominant infective strain. We report the structures of the Omicron spike trimer on its own and in complex with angiotensin-converting enzyme 2 (ACE2) or an anti-Omicron antibody. Most Omicron mutations are located on the surface of the spike protein and change binding epitopes to many current antibodies. In the ACE2-binding site, compensating mutations strengthen receptor binding domain (RBD) binding to ACE2. Both the RBD and the apo form of the Omicron spike trimer are thermodynamically unstable. An unusual RBD-RBD interaction in the ACE2-spike complex supports the open conformation and further reinforces ACE2 binding to the spike trimer. A broad-spectrum therapeutic antibody, JMB2002, which has completed a phase 1 clinical trial, maintains neutralizing activity against Omicron. JMB2002 binds to RBD differently from other characterized antibodies and inhibits ACE2 binding.

A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease-2019 (COVID-19), interacts with the host cell receptor angiotensin-converting enzyme 2 (hACE2) via its spike 1 protein during infection. After the virus sequence was published, we identified two potent antibodies against the SARS-CoV-2 receptor binding domain (RBD) from antibody libraries using a phage-to-yeast (PtY) display platform in only 10 days. Our lead antibody JMB2002, now in a Phase 1 clinical trial (ChiCTR2100042150), showed broad-spectrum in vitro blocking activity against hACE2 binding to the RBD of multiple SARS-CoV-2 variants, including B.1.351 that was reportedly much more resistant to neutralization by convalescent plasma, vaccine sera and some clinical-stage neutralizing antibodies. Furthermore, JMB2002 has demonstrated complete prophylactic and potent therapeutic efficacy in a rhesus macaque disease model. Prophylactic and therapeutic countermeasure intervention of SARS-CoV-2 using JMB2002 would likely slow down the transmission of currently emerged SARS-CoV-2 variants and result in more efficient control of the COVID-19 pandemic.

Expression and identification of a thermostable malate dehydrogenase from multicellular prokaryote Streptomyces avermitilis MA-4680.

A malate dehydrogenase (MDH) from Streptomyces avermitilis MA-4680 (SaMDH) has been expressed and purified as a fusion protein. The molecular mass of SaMDH is about 35 kDa determined by SDS-PAGE. The recombinant SaMDH has a maximum activity at pH 8.0. The enzyme shows the optimal temperature around 42 °C and displays a half-life (t(1/2)) of 160 min at 50°C which is more thermostable than reported MDHs from most bacteria and fungi. The k(cat) value of SaMDH is about 240-fold of that for malate oxidation. In addition, the k(cat)/K(m) ratio shows that SaMDH has about 1,246-fold preference for oxaloacetate (OAA) reduction over L-malate oxidation. The recombinant SaMDH may also use NADPH as a cofactor although it is a highly NAD(H)-specific enzyme. There was no activity detected when malate and NADP(+) were used as substrates. Substrate inhibition studies show that SaMDH activity is strongly inhibited by excess OAA with NADH, but is not sensitive to excess L-malate. Enzymatic activity is enhanced by the addition of Na(+), NH(4)(+), Ca(2+), Cu(2+) and Mg(2+) and inhibited by addition of Hg(2+) and Zn(2+). MDH is widely used in coenzyme regeneration, antigen immunoassays and bioreactors. The enzymatic analysis could provide the important basic knowledge for its utilizations.

Identification and biochemical characterization of a thermostable malate dehydrogenase from the mesophile Streptomyces coelicolor A3(2).

We identified and characterized a malate dehydrogenase from Streptomyces coelicolor A3(2) (ScMDH). The molecular mass of ScMDH was 73,353.5 Da with two 36,675.0 Da subunits as analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The detailed kinetic parameters of recombinant ScMDH are reported here. Heat inactivation studies showed that ScMDH was more thermostable than most MDHs from other organisms, except for a few extremely thermophile bacteria. Recombinant ScMDH was highly NAD(+)-specific and displayed about 400-fold (k(cat)) and 1,050-fold (k(cat)/K(m)) preferences for oxaloacetate reduction over malate oxidation. Substrate inhibition studies showed that ScMDH activity was inhibited by excess oxaloacetate (K(i)=5.8 mM) and excess L-malate (K(i)=12.8 mM). Moreover, ScMDH activity was not affected by most metal ions, but was strongly inhibited by Fe(2+) and Zn(2+). Taken together, our findings indicate that ScMDH is significantly thermostable and presents a remarkably high catalytic efficiency for malate synthesis.